JP6247302B2 - Identification of patients with abnormal fractional shortening - Google Patents

Description

  The present invention relates to a method for assessing whether a subject should be subjected to diagnostic assessment based on imaging. The method includes determining the amount (s) of cardiac troponin and / or fibroblast growth factor 23 (FGF-23) in a sample from a subject, and the amount (s) and reference amount thus determined. Based on comparison (s). The present invention also relates to a system for performing an assessment of whether a subject should be subjected to an imaging-based diagnostic assessment, and reagents and kits used in performing the methods disclosed herein. . Furthermore, the invention relates to a method for predicting the risk of death and / or cardiovascular events. Also included are methods for diagnosing early stage LVH in a subject with retained left ventricular ejection.

  Abnormal midwall shortening rate (MFS) indicates abnormal contraction and / or dilated ventricular function in the early preclinical stage during progression to left ventricular hypertrophy (LVH) and heart failure, and in myocardial ischemia . MFS is a measure of contractile function that identifies hypertensive patients with evidence of target organ damage, contractile reserve damage, and increased mortality. Therefore, identification of subjects with abnormal shortening rates is important. In particular, this identification allows appropriate prophylactic treatment to be initiated before left ventricular hypertrophy or even irreversible progression of heart failure occurs.

  The shortening rate is recognizable by using an image format (eg, Honda T et al. J Cardiol. 2002 Mar; 39 (3): 141-50 .; Palmiero P et al. Echocardiography. 2008 Jan; 25 {l): 20- 6). Shimizu G et al. Circulation. 1985; 71: 266-272). However, abnormal medial wall shortening rates are often not tested or missed. This is especially true for the elderly. Evaluation of shortening rates using echocardiography requires expensive instrumentation, specialized imaging techniques, and specialized image recording and interpretation skills. The widespread application of echocardiographic screening for abnormal MFS has been limited by considering cost versus cost. However, this inconvenience could be overcome using blood biomarkers related to the medial wall shortening rate.

Furthermore, biomarker based methods may be useful for stratifying patients for subsequent imaging techniques such as echocardiography or magnetic resonance imaging.
WO 2012/025355 describes a method for diagnosing cardiac dysfunction and / or structural abnormalities preceding heart failure by determining the amount of cardiac troponin and IGFBP7 in a subject carrying risk factors for developing heart failure. To do. The document discloses that a level of troponin T equal to or higher than 3.5 pg / ml is an indicator of cardiac dysfunction and / or structural abnormalities that precede heart failure. Subjects with <15% medium wall shortening rate (MFS) are not listed.

  Saytan et al. 2007 (Am J Kidney Dis. December; 50 (6): 1009-1019), a low medial wall shortening rate is a measure of poor contractile function and low medial wall in asymptomatic hemodialysis patients It is disclosed that the shortening rate correlates independently with log NT-proBNP (p <0.01). Furthermore, there was no significant correlation between cTnT and mWFS (p = 0.51).

  Kitagawa et al 2011 (Nephron Extra 1 (1): 166-177) discloses that BNP as well as hs troponin I can be used to detect left ventricular diastolic dysfunction in patients with non-diabetic chronic kidney disease.

  Fibroblast growth factor-23 (FGF-23) is a 32 kDa hormone that is secreted into the blood from bone cells. Its two functions are to induce urinary phosphite excretion and inhibit vitamin D activation; both effects occur in the renal proximal tubule. High concentrations of circulating FGF-23 have been found in patients with end-stage renal disease. The hormone is a key factor in the control of calcium-phosphate and vitamin D metabolism and has a causative role in the pathogenesis of LV hypertrophy, a major determinant of cardiovascular events.

  Kirkpantur et al 2011 (Nephrol Dial Transplant 26: 1346-1354) discloses that FGF-23 levels are associated with increased left ventricular mass index (LVMI) and myocardial performance index in dialysis patients (see Table 2). Wanna) No subjects with an abnormal shortening rate of <15% were examined (see Table 2; all subjects have FS = almost 30% shortening rate).

  Mirzaa et al. 2009 (Mirzaa Atherosclerosis 207, 546-551) is associated with elevated serum FGF-23 levels, and increased left ventricular weight, left ventricular hypertrophy and left ventricular geometry, and risk for the presence of left ventricular hypertrophy in older subjects Disclose a relationship between the increase in the blood pressure, which is more pronounced in patients with renal disease (eGFR <60 mL / min).

  Faul et al 2011 (J Clin Invest 121 (11): 4393-4408) investigated the causative role of FGF-23 in the pathogenesis of LVH in animal models, and chronically elevated FGF-23 levels Suggests a direct contribution to high rates of left ventricular hypertrophy and death. The document also discloses an association between elevated serum FGF-23 levels in humans and the prevalence of left ventricular hypertrophy, and in humans with chronic kidney disease, increased FGF-23 levels are associated with left ventricular hypertrophy It also discloses that it precedes the development of. Also described is a method for predicting the risk of a patient suffering from left ventricular hypertrophy in the future based on the detection of FGF-23.

  Gutierrez et al. 2009 (Circulation May 19; 119 (19): 2545-2552) discloses that FGF-23 concentrations are significantly associated with increased LVMI. An increase in log FGF-23 concentration was also significantly associated with the presence of left ventricular hypertrophy.

  Taddei et al 2011 (Heart Fail Rev 16: 615-620) discloses vitamin D deficiency, ie, reduced vitamin D levels, as a risk factor for LVH in patients with chronic kidney disease.

  Palmiero et al. 2008 (Echocardiography Jan; 25 (1): 20-6) show that the medial shortening rate is an important early sign of ventricular dysfunction in hypertensive patients even when diastolic function is normal Is disclosed.

Ix et al 2012 (JA Am College of Cardiol.) Discloses that FGF-23 can be used as a predictor in population subjects.
The IGFBP system plays an important role in cell proliferation and differentiation. IGF binding protein 7 (= IGFBP-7) is a 30 kDa modular glycoprotein known to be secreted by endothelial cells, vascular smooth muscle cells, fibroblasts, and epithelial cells (Ono, Y. et al., Biochem Biophys Res Comm 202 (1994) 1490-1496). In the literature, this molecule is also referred to as FSTL2; IBP 7; IGF binding protein related protein I; IGFBP7; IGFBP7v; IGFBP rP1; IGFBP7; IGFBPRP1; MAC25 protein; PGI2 stimulating factor; and PSF or prostacyclin stimulating factor. Low levels of IGFB-7 have been detected in random human sera, and increased serum levels have been shown to be associated with insulin resistance (Lopez-Bermejo, A. et al., J. Clinical Endocrinology). and Metabolism 88 (2003) 3401-3408, Lopez-Bermejo, A. et al., Diabetes 55 (2006) 2333-2339).

  US 2010/0285491 discloses the use of IGFBP-7 in the assessment of heart failure. The document further discloses that the marker, IGFBP-7, can be used to select treatment regimens for patients suffering from HF.

  Motiwala et al. Disclose that continuous measurement of IGFBP7 can be used as a predictor in patients with left ventricular systolic dysfunction (JACC, 2013, 61 (10), E565).

  Mimecan (often referred to as “osteoglycine”) is a multifunctional component of the extracellular matrix. Mimecan has been shown to be involved in the control of collagen fibril formation, a process that is essential in development, tissue repair, and metastasis (Tasheva et al., Mol. Vis. 8 (2002) 407-415). It plays a role in bone formation in combination with TGF-beta-1 or TGF-beta-2. Transcriptome analysis in rat and human heart tissue revealed a high correlation between mimecan and left ventricular weight and extracellular remodeling in dilated cardiomyopathy (Petretto, E. et al., Nature Genetics 40 (2008)). 546-552).

WO2011 / 012268 discloses that mimecan can be used as a marker for heart failure assessment.
Endostatin was originally isolated from murine hemangioendothelioma as a 20 kDa proteolytic fragment of type XVIII collagen (O'Reilly, MS et al., Cell 88 (1997) 277-285). Collagen represents an extracellular matrix protein family with a characteristic triple helix conformation that forms supramolecular aggregates, which play a major role in maintaining tissue structural integrity. Excessive collagen deposition leads to fibrosis that destroys the normal function of surrounding tissues. Endostatin is released from the alpha 1 chain of collagen XVIII by the action of various proteolytic enzymes (Ortega, N. and Werb, Z., Journal of Cell Science 115 (2002) 4201-4214). Endostatin is a potent inhibitor of angiogenesis and blood vessel growth.

WO2010 / 1224821 discloses that endostatin can be used as a marker for heart failure assessment.
The technical problem underlying the present invention can be seen as providing means and methods to meet the aforementioned needs.

  The technical problem is solved by the embodiments characterized in the claims and herein below.

A method for evaluating whether a subject should be subjected to diagnostic evaluation based on imaging method The present invention is a method for evaluating whether a subject should be subjected to diagnostic evaluation based on imaging method,
a) determining in the sample from the subject the amount (s) of cardiac troponin and / or fibroblast growth factor 23 (FGF-23) and b) referring to the amount (s) thus determined Comparing the amount (s), thereby assessing whether the subject should be subjected to imaging-based diagnostic evaluation;
Relates to the method comprising the steps of:

  Preferably, the subject is imaged by performing further step c), which evaluates whether the subject should be subjected to an imaging-based diagnostic evaluation based on the results of the comparison performed in step b). Assess whether it should be used for diagnostic evaluation based on

  The method of the present invention is preferably an ex vivo or in vitro method. Further, the method may include steps in addition to those explicitly mentioned above. For example, further steps may relate to sample pretreatment or evaluation of results obtained by the method. The method may be performed manually or the method may be assisted by automation. Preferably, steps (a) and / or (b) are performed in whole or in part by automation, for example for determination in step (a) or in a computer-implemented comparison in step (b) and / or said It may be assisted by a suitable robot and sensor device for comparison-based evaluation.

Accordingly, the present invention is also preferably a system for assessing whether a subject should be subjected to an imaging based diagnostic assessment comprising:
a) set in contact with a portion of the sample from the subject in vitro with a ligand comprising specific binding affinity for cardiac troponin and / or with a ligand comprising specific binding affinity for FGF-23; Analyzer unit,
b) an analyzer unit configured to detect a signal from a portion of a sample from a subject that has been contacted with the ligand (s),
c) a computing device having a processor and in communication with the analyzer apparatus; and d) a non-transitory machine readable medium comprising a plurality of instructions executable by the processor, the instructions comprising: When performed, the amount (s) of cardiac troponin and / or FGF-23 is calculated and the reference amount (s) is compared with the amount (s), thereby determining the subject. It relates to said system comprising said medium for evaluating whether it should be subjected to diagnostic evaluation based on imaging methods.

FIG. 1A: Endostatin and NT-proBNP levels in female subjects with borderline LVH / mildly elevated LV weight. FIG. 1B: Endostatin and cTnT levels in female subjects with borderline LVH / mildly elevated LV weight. FIG. 2A: Endostatin and cTnT levels in male subjects with borderline LVH / mildly elevated LV weight. FIG. 2B: Endostatin and NT-proBNP levels in male subjects with borderline LVH / mildly elevated LV weight.

  The term “assessing” as used herein means determining whether a subject as set forth herein should be subjected to imaging-based diagnostic evaluation. Therefore, by performing the method of the present invention, it is possible to stratify whether or not a subject as shown herein should be subjected to diagnostic assessment based on imaging. By performing the methods of the invention, it is also possible to identify subjects that should not be subjected to these diagnostic assessments, for example. These assessments require expensive instrumentation, specialized imaging techniques, and specialized image recording and interpretation skills, thus avoiding unnecessary medical costs. A subject that is sensitive to imaging-based assessment is preferably a subject that has an increased likelihood of suffering from systolic and / or diastolic dysfunction. The subject therefore requires diagnostic assessment based on imaging. In particular, a subject that is sensitive to assessment based on imaging is preferably a subject that has an increased likelihood of suffering from an abnormal medial wall shortening rate. Subjects that are not sensitive to imaging-based assessment are preferably subjects that have a reduced likelihood of suffering from systolic and / or diastolic dysfunction. Thus, the subject does not require diagnostic assessment based on imaging methods. In particular, subjects that are not sensitive to imaging-based assessment are preferably subjects that have a reduced likelihood of suffering from an abnormal medial wall shortening rate.

  As will be appreciated by those skilled in the art, such an assessment is usually not intended to be accurate for 100% of the subjects to be assessed. The term, however, requires that a statistically significant portion of the subject can be accurately assessed. Whether or not the evaluation is correct can be confirmed by a method well known in the art. Furthermore, whether a portion is statistically significant can be determined by those skilled in the art using various well-known statistical evaluation tools such as confidence interval determination, p-value determination, Student's t-test, Mann-Whitney test, etc. It can be easily determined. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001.

  According to the method of the present invention, it shall be evaluated whether the subject to be tested should be subjected to diagnostic evaluation based on imaging. Preferably, diagnostic assessment based on imaging is aimed at generating a picture of the heart. Therefore, the evaluation shall be an evaluation of the heart. More preferably, the diagnostic assessment based on imaging is echocardiography or magnetic resonance imaging (MRI). The most preferred diagnostic assessment is echocardiography.

  The imaging techniques described above are well known in the art. The term “echocardiology” as used herein preferably refers to the assessment of cardiac structure and function using images and recordings produced by ultrasound. Preferably, the term includes one-dimensional, two-dimensional and three-dimensional echocardiography. Further, the term “echocardiography” is intended to include transthoracic echocardiography, stress echocardiography, and contrast echocardiography. In a particularly preferred embodiment, the echocardiogram is an M mode echocardiogram.

  Magnetic resonance imaging is a medical imaging technique used in radiology to visualize the internal structure of the body in detail. When high frequency radio waves irradiate water atoms and hydrogen atoms in other tissue components placed in a strong magnetic field, the absorption and transmission of the radio waves are analyzed by MRI. The term “magnetic resonance imaging” as used herein refers to cardiac magnetic resonance imaging (often referred to as cardiovascular magnetic resonance imaging).

  In a preferred embodiment of the method of the invention, diagnostic assessment based on imaging is used to diagnose diastolic dysfunction. In an even more preferred embodiment of the method of the invention, imaging-based diagnostic evaluation is used to diagnose systolic dysfunction.

  The term “diastolic dysfunction” is well understood by those skilled in the art. Preferably, the term refers to a decrease in cardiac pump function due to ventricular filling disorder. Thus, the term preferably refers to a decrease in the performance of one or both ventricles of the heart during diastole. The term “systolic dysfunction” is also well known in the art. As used herein, the term preferably refers to a decrease in cardiac pump function due to a decrease in ventricular contraction. Preferably, systolic and / or diastolic dysfunction is not accompanied by overt signs of heart failure. Again preferably, systolic and / or diastolic dysfunction is not accompanied by left ventricular hypertrophy.

In a particularly preferred embodiment of the method of the present invention, diagnostic evaluation based on imaging is used to diagnose an abnormal medial wall shortening rate, particularly an abnormal left ventricular medial wall shortening rate.
The expression “medium wall shortening rate” is well known in the art (abbreviated herein as “MFS”). It is well known in the art that reduced left ventricular (LV) MFS is an early sign of LV dysfunction. Preferably, the medial wall shortening rate is considered abnormal if it is lower than 15%. This cut-off point has been used as a reference value in the setting of HF, and a prognostic association in hypertensive subjects has been demonstrated (Murredu et al., European Journal of Failure (2012) 14, 718-729). See). Again preferably, the medial wall shortening rate is considered abnormal if it is lower than 14% or 13%. Furthermore, if the medial wall shortening rate is higher than (or equal to) 15%, it is considered normal. Again preferably, the medial wall shortening rate is considered normal if it is higher than 16% or 17%.

  The medium wall shortening rate can be calculated by a known method. For example, for its entire disclosure, it can be calculated from a two-shell cylindrical model as described by Shimizu et al. (Shimizu G, Hirota Y, Kita Y, Kawamura, K, Saito T Gaach WH, left ventricular medial wall dynamics in systemic arterial hypertension Circulation 1991; 83: 1676-84). This method is a refinement of the conventional medial wall method and provides data reflecting the shortening of the theoretical surrounding medial wall fibers or myocardial rings. The method assumes that the left ventricular weight is constant throughout the cardiac cycle and does not require the assumption that the inner and outer wall thickening rates are equal.

  The determination of MFS is also described by Mayet et al. Or in a previous article by Shimizu et al., All of which are incorporated herein (eg, Mayet et al., Hypertension. 2000; 36: 755-759; Shimizu G , Zile MR, Blastein AS, Gaash WH, left ventricular filling and medial wall fiber elongation in patients with left ventricular hypertrophy: overestimation of fiber velocity by conventional medial wall measurements Circulation. Shimizu G, Conrad CH, Gaash WH.Phase plane analysis of left ventricular filling and midwall fiber elongation in patients with left ventricular hypertrophy Circulation. 1987; 75 (suppl l): I-34-I-39 It is to be irradiation). Furthermore, the determination of MFS has been described by de Simone et al. (JACC, 1994, Vol. 23 (6): 1444-51), which is also incorporated herein in its entirety. Preferably, MFS is determined herein according to the method as described by Shimizu in 1987 and 1985, more preferably MFS is determined according to Mayet et al., Most preferably MFS is determined according to de Simone.

  The term “subject” as used herein relates to animals, preferably mammals, and more preferably humans. A subject in the context of the present invention may be male or female. Furthermore, it is specifically envisaged that the subject is older than 65 years.

In a preferred embodiment of the invention, the subject does not suffer from left ventricular hypertrophy. In particular, it is specifically envisaged that the subject has a normal left ventricular weight.
The term “left ventricular hypertrophy” is well known in the art. A detailed review on left ventricular hypertrophy can be found, for example, in standard textbooks (see Swami Curr Cardio Rep (2010) 12: 277-282). LVH can be detected by electrocardiogram, echocardiography, or cardiac magnetic resonance imaging (MRI). Preferably, LVH is detected by echocardiography. Furthermore, criteria for LVH diagnosis are well known in the art (Mancia et al., European Heart J. 2007, 28: 1462, Die Inner Medizin: Referenzfurk fur den Challage-Wolfgur 7Wolf gen. Rep (2010) 12: 277-282).

  Assessment of LVH preferably includes measurements of septal wall diameter, left ventricular posterior wall thickness, and diastolic diameter, along with calculations of left ventricular weight according to formulas known in the art. Particularly preferred criteria for diagnosing LVH are disclosed, for example, in guidelines (Mancia et al., European Heart J. 2007, 28: 1462). Also preferably, the criteria may be taken from ACC / AHA guidelines for the assessment and management of chronic heart failure in adults. J. et al. Am. Coll. Cardiol. 2001; 38; 2101-2113.

  Preferably, the Cornell voltage criterion, the Cornell product criterion, the Sokolow-Lyon voltage criterion or the Romhilt-Etes point score system is used (Mancia et al., European Heart J. 2007, 28: 1462).

  The term “left ventricular hypertrophy” (abbreviated “LVH”) is preferably referred to herein as thickening of the ventricular wall. LVH is preferably a response to a chronically increased workload on the heart. LVH, found in patients with arterial hypertension (ie high blood pressure), is a disease that requires treatment. In the context of the present invention, cardiac functional and / or structural abnormalities typically precede left ventricular hypertrophy and / or heart failure.

  Hypertrophy as a response to increased afterload associated with increased systemic vascular resistance is necessary to a specific point and is protective. Beyond that point, various dysfunctions are associated with LVH, including lower coronary vasodilatation, suppressed left ventricular wall dynamics, and abnormal left ventricular diastolic filling patterns.

Preferably, the male subject has a left ventricular weight index (and thus a ratio of left ventricular weight to body surface abbreviated LVMI) of greater than 105 g / m ² , more preferably greater than 110 g / m ² , and Most preferably, if it is higher than 115 g / m ² (or especially higher than 116 g / m ² ), it suffers from LVH. Preferably, the female subject suffers from LVH if the LVMI is higher than 85 g / m ² , even more preferably higher than 90 g / m ² and most preferably higher than 96 g / m ² .

As described herein above, the subject preferably has a normal left ventricular weight. If the subject is male, the following applies: The subject preferably has a male subject's left ventricular weight index equal to or lower than 105 g / m ² , or more preferably equal to 110 g / m ² . Or lower, or most preferably equal to or lower than 115 g / m ² (or in particular equal to or lower than 116 g / m ² ), it is considered to have a normal left ventricular weight. When the subject is female, the following applies: The subject preferably has a subject's left ventricular weight index preferably equal to or lower than 85 g / m ² , or more preferably equal to 90 g / m ² If it is or lower, or most preferably equal to or lower than 96 g / m ² , it is considered to have a normal left ventricular weight. It should be understood that subjects with normal left ventricular weight do not suffer from LVH.

  Preferably, LVMI is determined as disclosed by Lang et al. (Recommendation for ventricular quantification Eur J Echocardiogr 2006; 7: 79-108).

  In a preferred embodiment of the invention, the subject suffers from hypertension. The hypertension may be any type of hypertension known to those skilled in the art. In a preferred embodiment of the invention, the individual suffers from arterial hypertension, systolic hypertension and / or diastolic hypertension. In particular, the subject shall suffer from arterial hypertension. Preferably, the subject suffers from arterial hypertension if the systolic pressure exceeds 140 mmHg and / or the diastolic pressure exceeds 90 mmHg. More preferably, the subject is 3 months or longer, 6 months or longer, 12 months or longer, 2 years or longer, 3 years or longer, or 5 years or longer. If the systolic pressure exceeds 140 mmHg and / or the diastolic pressure exceeds 90 mmHg, then arterial hypertension is suffered. Preferably, blood pressure temporarily elevated, for example by exercise, is not included in the term “arterial hypertension”.

Hypertension may be accompanied by risk factors that develop heart failure. Preferred risk factors for developing heart failure are, for example, asymptomatic organ damage of the heart, brain, kidneys, blood vessels; obesity, preferably obesity defined by body mass index BMI <25 kg / m ² ; adiposity; metabolic syndrome; 1 Type 2 or type 2 diabetes, especially type 2 diabetes; type 1 diabetes with microalbuminuria, smoking; history of vascular reconstruction; history of cardiotoxic medication or alcohol abuse; dyslipidemia; total cholesterol, total cholesterol / HDL-cholesterol ratio , Personal history of rheumatic fever; family history of cardiomyopathy. Thus, a subject may also suffer from hypertension associated with one or more of the additional risk factors described above. However, it is also contemplated that the subject suffers from hypertension only.

  It is further envisaged that the subject does not suffer from ACS (acute coronary syndrome). The term “ACS” as used herein includes STEMI (ST elevation myocardial infarction), NSTEMI (non-ST elevation myocardial infarction) and unstable angina. It is further envisioned that the subject to be tested has no ACS history. In particular, the subject shall not suffer from ACS within one month of performing the method of the present invention (more precisely, within one month prior to obtaining the sample).

  It is also contemplated that the subject does not suffer from coronary artery disease. The term “coronary artery disease”, abbreviated CAD, is often referred to as coronary heart disease (CHD) or atherosclerotic heart disease and is known to those skilled in the art. Preferably, the term refers to a condition in which a blood vessel supplying blood and oxygen to the heart is stenotic. Coronary artery disease is usually caused by a condition called atherosclerosis, which occurs when fatty components and substances called plaques accumulate on the arterial wall. This narrows the blood vessel. In particular, CAD is the result of the accumulation of atherosclerotic plaque within the arterial wall that feeds the heart muscle (heart muscle). Preferably, a subject with CAD has at least 50% stenosis (and thus at least 50% occlusion) in at least one major coronary artery. Thus, a subject not suffering from CAD preferably has less than 50% stenosis in the main coronary artery. Methods of assessing the degree of coronary occlusion are well known in the art, and preferably the degree is assessed by coronary angiography. Symptoms and signs of coronary artery disease are observed in the advanced state of the disease, but most patients with coronary artery disease have a disease until the first onset of an acute event, often a “sudden” heart attack, eventually occurs. As it progresses, it shows no evidence of disease for decades.

Further, the subject shall not be pregnant. Also, the subject is preferably not an athlete.
It is further envisaged that the subject does not suffer from heart failure and / or does not show obvious symptoms of heart failure.

  The term “heart failure” herein relates to impaired contractile and / or diastolic functions, preferably accompanied by overt signs of heart failure, as known to those skilled in the art. Preferably, the heart failure referred to herein is also chronic heart failure. Heart failure according to the present invention includes overt and / or advanced heart failure. In overt heart failure, the subject exhibits symptoms of heart failure as known to those skilled in the art. The term “heart failure” is specifically intended herein to refer to stages C and D of the ACC / AHA classification. In these stages, the subject exhibits typical symptoms of heart failure, i.e., the subject is not apparently healthy. Subjects who have heart failure and are classified as stage C or D have permanent, irreversible structural and / or functional changes to the myocardium, and these changes result in complete Healthy recovery is impossible.

  The ACC / AHA classification is a classification of heart failure developed by the American Heart Association and the American Heart Association (see J. Am. Coll. Cardiol. 2001; 38; 2101-2213, 2005 revision. J. Am. Coll. Cardiol. 2005; 46; see e1-e82). Four stages are defined, A, B, C and D. Stages A and B are not HF (heart failure), but are considered to help identify patients early before developing “true” HF. Stage A and B patients are best defined as patients with risk factors for developing HF. For example, a patient with coronary artery disease, hypertension, or diabetes that still does not exhibit left ventricular (LV) dysfunction, hypertrophy, or geometric ventricular deformity will be considered stage A, while asymptomatic. However, patients exhibiting LV hypertrophy (LVH, a phenomenon in which the ventricular wall thickens) and / or LV dysfunction will be designated as stage B. Stage C then shows patients with the current or past symptoms of HF associated with the underlying structural heart disease (the majority of HF patients), and stage D shows the true refractory HF. Specify a patient.

  In a preferred embodiment of the invention, the subject to be tested is classified as a stage B subject (according to the ACC / AHA classification as described above). Thus, the subject does not show obvious signs of heart failure. When a subject is classified as a Stage B subject, the subject preferably has a normal left ventricular weight, as specified in more detail elsewhere herein.

  Furthermore, in the context of the present invention, it is envisaged that the subject has no renal dysfunction. Preferably, the subject is not suffering from renal failure, in particular the subject is not suffering from acute, chronic and / or end stage renal failure. Further, the subject preferably does not suffer from renal hypertension. Methods for assessing whether a subject exhibits impaired renal function are well known in the art. It is also possible to diagnose kidney damage by any means known and deemed appropriate. In particular, renal function can be evaluated by glomerular filtration rate (GFR). For example, the GFR can also be calculated by the Cockgroft-Gault or MDRD formula (Levey 1999, Anals of Internal Medicine, 461-470). GFR is the volume of fluid that is filtered from the glomerular capillaries into the Bowman's sac per unit time. Clinically, this is often used to determine renal function. GFR was originally estimated by injecting insulin into the plasma (GFR is not determinable, and all calculations obtained from formulas such as the Cockroft-Gault formula or MDRD formula describe the estimate And not “true” GFR). Since insulin is not reabsorbed by the kidney after glomerular filtration, its excretion rate is directly proportional to the water and solute filtration rate of the glomerular filter. However, in clinical practice, creatinine clearance is used to measure GFR. Creatinine is an endogenous molecule, synthesized in the body and freely filtered by the glomeruli (but also very small amounts are secreted by the renal tubules). Creatinine clearance (CrCl) is therefore an approximation of GFR. GFR is typically recorded in milliliters per minute (mL / min). The normal range for GFR for men is 97-137 mL / min, and the normal range for GFR for women is 88-128 ml / min. Thus, it is specifically contemplated that the GFR of subjects who do not exhibit impaired renal function is within this range. In addition, the subject preferably has a blood creatinine level (particularly serum creatinine level lower than 0.9 mg / dl, more preferably lower than 1.1 mg / dl, and most preferably lower than 1.3 mg / dl). ).

If the method of the present invention involves determining the amount of FGF-23 (and vitamin D), the subject should preferably not exhibit vitamin D deficiency.
Further, in the context of a method for assessing whether a subject should be subjected to imaging-based diagnostic assessment, a method for diagnosing abnormal MFS, and a method for predicting the risk of death, the subject Preferably do not have increased natriuretic peptide levels. Preferably, the subject does not have increased brain natriuretic peptide levels. More preferably, the subject does not have increased BNP-type natriuretic peptide levels. Most preferably, the subject does not have increased NT-proBNP levels. Preferably, the subject has a NT-proBNP level lower than 125 pg / ml for <75 years old, and> 450 pg / ml for <75 years, or more preferably, particularly in blood, serum or plasma samples Has an NT-proBNP level lower than 100 pg / ml for <75 years old, and> 300 pg / ml for ≧ 75 years old.

  The term “sample” refers to a body fluid sample, a sample of separated cells, or a sample from a tissue or organ. The bodily fluid sample can also be obtained by well-known techniques and preferably includes a blood, plasma, serum or urine sample, more preferably a blood, plasma or serum sample. A tissue or organ sample can be obtained from any tissue or organ, for example, by biopsy. Separated cells can be obtained from body fluids, or tissues or organs by separation techniques such as centrifugation or cell sorting. Preferably, the cell, tissue or organ sample is obtained from a cell, tissue or organ that expresses or produces a peptide referred to herein.

  The term “cardiac troponin” refers to all troponin isoforms expressed in cardiac cells, and preferably subendocardial cells. These isoforms are described, for example, in Anderson 1995, Circulation Research, vol. 76, no. 4: 681-686 and Ferrieres 1998, Clinical Chemistry, 44: 487-493, which are well characterized in the art. Preferably, the cardiac troponin is troponin T and / or troponin I, and most preferably troponin T. It is understood that troponin isoforms can also be determined in the method of the invention together, i.e. simultaneously or sequentially, or individually, i.e. without determining any other isoforms. Shall. The amino acid sequences of human troponin T and human troponin I are disclosed in Anderson, ibid. And Ferrieres 1998, Clinical Chemistry, 44: 487-493.

  The term “cardiac troponin” also includes variants of the aforementioned specific troponins, ie preferably troponin I, and more preferably troponin T. Such variants have at least the same essential biological and immunological properties as the particular cardiac troponin. In particular, they are of the same essential nature if they are detectable by the same specific assay as mentioned herein, eg by an ELISA assay using polyclonal or monoclonal antibodies that specifically recognize the cardiac troponin. Share biological and immunological properties. Furthermore, a variant as referred to in accordance with the present invention is understood to have a different amino acid sequence due to at least one amino acid substitution, deletion and / or addition, wherein the amino acid sequence of the variant is still Preferably at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about the amino acid sequence of a particular troponin (especially over the entire length) About 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identical. Preferably, the degree of identity shall be determined by comparing two optimally aligned sequences over a comparison window, where fragments of amino acid sequences in the comparison window are for optimal alignment. , May include additions or deletions (eg, gaps or overhangs) when compared to a reference sequence (not including additions or deletions). Determine the number of positions where the same amino acid residue is present in both sequences to obtain the number of matched positions, divide the number of matched positions by the total number of positions in the comparison window, and add 100 to the result. Calculate the percentage by multiplying by to get the percent sequence identity. Optimal alignment of sequences for comparison is described by Smith and Waterman Add. APL. Math. 2: 482 (1981), according to the Needleman and Wunsch J. et al. Mol. Biol. 48: 443 (1970) by the homology alignment algorithm of Pearson and Lipman Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988) by a computerized implementation of these algorithms (GAP, BESTFIT, BLAST, PASTA, and TFSTA, Genetics Computer Group (GCG) in Wisconsin Genetics Software Package (75) Science Dr., Madison, Wis.) Or by visual inspection. If two sequences are identified for comparison, preferably GAP and BESTFIT are used to determine the optimal alignment and therefore the degree of identity. Preferably, default values of 5.00 for gap weight and 0.30 for gap weight length are used. The variant may be an allelic variant or any other species-specific homologue, paralog, or ortholog. Furthermore, variants referred to herein include specific cardiac troponins or fragments of the aforementioned types of variants, so long as the fragments have essential immunological and biological properties as described above. . Preferably, the cardiac troponin variant has immunological properties (ie, epitope composition) comparable to that of human troponin T or troponin I. Thus, the variant should be recognizable by the aforementioned means or ligands used to determine the concentration of cardiac troponin. Such a fragment may be, for example, a degradation product of troponin. Also included are variants that differ due to post-translational modifications such as phosphorylation or myristination. Preferably, the biological property of troponin I and variants thereof is the ability to inhibit actomyosin ATPase or to inhibit in vivo and in vitro angiogenesis, these are Moses et al. 1999 PNAS USA 96. (6): Detectable based on the assay described by 2645-2650. Preferably, the biological properties of troponin T and its variants form a complex with troponin C and I and bind to calcium ions, or preferably a complex of troponin C, I and T, or troponin When present as a complex formed by C, troponin I and troponin T variants, the ability to bind to tropomyosin. Low concentrations of circulating cardiac troponin are known to be detectable in subjects in a variety of conditions, but further research is needed to understand their role and rate (Masson et al., Curr Heart Fail). Rep (2010) 7: 15-21).

  Preferably, the cardiac troponin is troponin T, especially human troponin T. Preferably, the amount of troponin T is determined by using a sensitive troponin assay as described in the Examples or in WO2012 / 025355.

  Fibroblast growth factor-23 (abbreviated as “FGF-23”) is an important factor in the control of calcium phosphate and vitamin D metabolism and the pathogenesis of LV hypertrophy, an important determinant of cardiovascular events Has a causative role. Fibroblast growth factor-23 (FDF-23) is a 32 kDa hormone that is secreted from bone cells into the blood. Its two functions are to induce urinary phosphite excretion and inhibit vitamin D activation; both effects occur in the renal proximal tubule. High concentrations of circulating FGF-23 have been found in patients with end-stage renal disease. Preferably, FGF-23 is human FGF-23. The sequence of human FGF-23 is well known in the art, for example the amino acid sequence is accessible through GenBank accession number NM — 020638.1 GI: 10190673. Furthermore, the sequence is also described in Shimada et al., 2001, PNAS, vol. 98 (11) 6500-6505. The term “FGF-23” also includes variants of the aforementioned FGF-23. Such variants have at least the same essential biological and immunological properties as the specific FGF-23. In particular, they are of the same essential nature if they are detectable by the same specific assay as mentioned herein, for example by an ELISA assay using a polyclonal or monoclonal antibody that specifically recognizes said FGF-23. Share common biological and immunological properties. Furthermore, a variant as referred to in accordance with the present invention shall be understood to have a different amino acid sequence due to at least one amino acid substitution, deletion and / or addition, wherein the amino acids of the variant The sequence is still preferably at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, preferably with the amino acid sequence of FGF-23 (especially over the entire length). %, At least about 92%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identical. Methods for calculating the degree of identity are disclosed elsewhere herein. Preferably, FGF-23 is available from Immupics, Inc. Human FGF-23 (C-terminal) ELISA kit (eg, distributed under catalog number 60-6100).

  A peptide or polypeptide referred to herein is selected from the group consisting of cardiac troponin, fibroblast growth factor 23 (FGF-23), IGFBP7 (IGF binding protein 7), GDF-15, endostatin and mimecan, among others. The step of determining the amount of marker to be used relates to the step of measuring the amount or concentration, preferably semi-quantitatively or quantitatively. Measurements can also be made directly or indirectly. Direct measurement relates to measuring the amount or concentration of a peptide or polypeptide based on the signal obtained from the peptide or polypeptide itself and the intensity that directly correlates with the number of peptide molecules present in the sample. Such signals are sometimes referred to herein as intensity signals, and can be obtained, for example, by measuring intensity values of specific physical or chemical properties of a peptide or polypeptide. For indirect measurements, measure signals from secondary components (ie, components that are not peptides or polypeptides themselves) or biological readings, such as measurable cellular reactions, ligands, labels, or enzymatic reaction products The process of carrying out is included.

  According to the present invention, the step of determining the amount of peptide or polypeptide can be achieved by all known means for determining the amount of peptide in a sample. Such means include immunoassays and methods that can utilize the labeled molecule in a variety of sandwich, competition, or other assay formats. Such assays are preferably based on detection agents, such as antibodies that specifically recognize the peptide or polypeptide to be determined. The detection agent shall be capable of producing a signal indicating the presence or absence of the peptide or polypeptide, either directly or indirectly. Furthermore, the signal intensity can preferably be directly or indirectly (eg inversely proportional) to the amount of polypeptide present in the sample. Further suitable methods include measuring physical or chemical properties specific for the peptide or polypeptide, such as its exact molecular weight or NMR spectrum. The method preferably comprises a biosensor, an optical device coupled to an immunoassay, a biochip, an analytical device, such as a mass analyzer, an NMR analyzer, or a chromatography device. In addition, methods include microplate ELISA based methods, fully automated or robotic immunoassays (eg available on Elecsys ™ analyzer equipment), CBA (eg enzymatic cobalt binding assays available on Roche-Hitachi ™ analyzer equipment). , And latex agglutination assays (available on, for example, the Roche-Hitachi ™ analyzer device).

  Preferably, the step of determining the amount of peptide or polypeptide comprises: (a) a cell capable of inducing a cellular response whose intensity is indicative of the amount of peptide or polypeptide; Contacting for a period of time, and (b) measuring the cellular response. In order to measure the cellular response, the sample or processed sample is preferably added to the cell culture and the internal or external cell response is measured. A cellular response can include measurable expression of a reporter gene or secretion of a substance, such as a peptide, polypeptide, or small molecule. Expression or material should produce an intensity signal that correlates to the amount of peptide or polypeptide.

  Again preferably, determining the amount of peptide or polypeptide comprises measuring a specific intensity signal that can be obtained from the peptide or polypeptide in the sample. As noted above, such a signal can be the signal intensity observed in a peptide or polypeptide specific m / z variable observed in a peptide or polypeptide specific mass spectrum or NMR spectrum. is there.

  Determining the amount of peptide or polypeptide preferably comprises (a) contacting the peptide with a specific ligand, (b) (optionally) removing unbound ligand, and (c) bound ligand, ie, step It is also possible to include a step of measuring the amount of the ligand complex formed in (a). According to a preferred embodiment, the steps of contacting, removing and measuring may be performed by the analyzer unit of the system disclosed herein. In accordance with some aspects, the process can be performed by a single analyzer unit of the system or by more than one analyzer unit with communication capable of each other. For example, according to certain embodiments, the system disclosed herein includes a first analyzer unit for performing the contacting and removal steps, and a first analyzer unit by a transport device (eg, a robot arm). A second analyzer unit may be included that performs the step of measuring and is operably coupled to the second analyzer unit.

  The bound ligand, ie, the ligand or ligand / peptide complex will produce an intensity signal. Bonds according to the present invention include both covalent and non-covalent bonds. A ligand according to the present invention may be any compound that binds to a peptide or polypeptide described herein, such as a peptide, polypeptide, nucleic acid, or small molecule. Preferred ligands include antibodies, nucleic acids, peptides or polypeptides, such as receptors or binding partners for the peptides or polypeptides, and fragments thereof that contain a binding domain for the peptides, and aptamers, such as nucleic acids or peptide aptamers. Methods for preparing such ligands are well known in the art. For example, identification and production of suitable antibodies or aptamers are also provided by commercial suppliers. Those skilled in the art are well aware of how to develop derivatives of ligands with higher affinity or specificity. For example, random mutations can be introduced into nucleic acids, peptides or polypeptides. These derivatives can then be tested for binding according to screening methods known in the art, such as phage display. Antibodies as referred to herein include both polyclonal and monoclonal antibodies, as well as fragments thereof, such as Fv, Fab and F (ab) 2 fragments, capable of binding to an antigen or hapten. The present invention also includes single chain antibodies and humanized hybrid antibodies, where the amino acid sequence of a non-human donor antibody that exhibits the desired antigen specificity is combined with the sequence of a human acceptor antibody. The donor sequence usually includes at least the antigen binding amino acid residues of the donor, but may also include other structurally and / or functionally appropriate amino acid residues of the donor antibody. Such hybrids can be prepared by several methods well known in the art. Preferably, the ligand or agent specifically binds to the peptide or polypeptide. Specific binding in accordance with the present invention is such that the ligand or agent must not substantially bind ("cross-react" with) another peptide, polypeptide or substance present in the sample to be analyzed. Means. Preferably, the specifically binding peptide or polypeptide has an affinity that is at least 3 times higher, more preferably at least 10 times higher, and even more preferably at least 50 times higher than any other related peptide or polypeptide. Must be combined. Non-specific binding can be tolerated if it is still distinguishable and clearly measurable, for example according to size on the Western blot or by higher abundance in the sample. Binding of the ligand can be measured by any method known in the art. Preferably, the method is semi-quantitative or quantitative. Additional suitable techniques for polypeptide or peptide determination are described below.

It is also possible to measure the binding of the ligand directly, for example by NMR or surface plasmon resonance. According to a preferred embodiment, the measurement of ligand binding is performed by the analyzer unit of the system disclosed herein. The amount of binding measured can then be calculated by the computing device of the system disclosed herein. If the ligand also serves as a substrate for the enzymatic activity of the peptide or polypeptide of interest, the enzyme reaction product may be measured (eg, by measuring the amount of cleaved substrate, eg, on a Western blot). It is also possible to measure the amount of protease). Alternatively, the ligand can itself exhibit enzymatic properties and the “ligand / peptide or polypeptide” complex, or the ligand respectively bound by the peptide or polypeptide, can be contacted with an appropriate substrate. It is also possible to enable detection by generation of an intensity signal. For measurement of enzyme reaction products, preferably the amount of substrate is a saturating amount. The substrate may also be labeled with a detectable label prior to the reaction. Preferably, the sample is contacted with the substrate for an appropriate period. An appropriate period refers to the time required for a detectable, preferably measurable amount of product to be produced. Instead of measuring the amount of product, it is also possible to measure the time required for the appearance of a given (eg detectable) amount of product. Third, the ligand can be coupled, either covalently or non-covalently, to a label that allows detection and measurement of the ligand. Labeling may be done by direct or indirect methods. Direct labeling involves coupling the label directly (covalently or non-covalently) to the ligand. Indirect labeling involves binding (covalently or non-covalently) a secondary ligand to a first ligand. The secondary ligand must bind specifically to the first ligand. The secondary ligand may be coupled to a suitable label and / or the secondary ligand may be the target (receptor) of a tertiary ligand that binds to the secondary ligand. Secondary, tertiary, or even higher order ligands are often used to increase the signal. Other suitable secondary and higher ligands can also include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). The ligand or substrate may also be “tagged” with one or more tags as known in the art. Such tags can then be targets for higher order ligands. Suitable tags include biotin, digoxigenin, His-tag, glutathione-S-transferase, FLAG, GFP, myc-tag, influenza A virus hemagglutinin (HA), maltose binding protein, and the like. In the case of a peptide or polypeptide, the tag is preferably at the N-terminus and / or C-terminus. A suitable label is any label detectable by a suitable detection method. Typical labels include gold particles, latex beads, acridan esters, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels (eg “magnetic beads” including paramagnetic and superparamagnetic labels), and fluorescent labels Is included. Enzymatic activity labels include, for example, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, luciferase, and derivatives thereof. Suitable substrates for detection include diaminobenzidine (DAB), 3,3′-5,5′-tetramethylbenzidine, NBT-BCIP (4-nitroblue tetrazolium chloride, available as a ready-made stock solution from Roche Diagnostics, And 5-bromo-4-chloro-3-indolyl-phosphate), CDP-Star ^™ (Amersham Biosciences), ECF ^™ (Amersham Biosciences). Appropriate enzyme-substrate combinations can also produce colored reaction products, fluorescence or chemiluminescence, which are measured according to methods known in the art (eg, using a light-sensitive film or a suitable camera system). Is possible. For the measurement of enzymatic reactions, the above-mentioned criteria apply in the same way. Typical fluorescent labels include fluorescent proteins (eg, GFP and its derivatives), Cy3, Cy5, Texas Red, fluorescein, and Alexa dyes (eg, Alexa568). Additional fluorescent labels are available from, for example, Molecular Probes (Oregon). The use of quantum dots as fluorescent labels is also contemplated. Typical radioactive labels include 35S, 125I, 32P, 33P, and the like. The radioactive label can be detected by any known and appropriate method, such as a light-sensitive film or a phosphoimager. Suitable assays according to the invention also include precipitation (especially immunoprecipitation), electrochemiluminescence (electrogenerated chemiluminescence), RIA (radioimmunoassay), ELISA (enzyme linked immunosorbent assay), sandwich enzyme immunoassay, Includes electrochemiluminescence sandwich immunoassay (ECLIA), dissociation enhanced lanthanide fluorescence immunoassay (DELFIA), scintillation proximity assay (SPA), turbidimetry, turbidimetry, latex enhanced turbidimetry or turbidimetry, or solid phase immunoassay It is. Additional methods known in the art (eg, gel electrophoresis, 2D gel electrophoresis, SDS polyacrylamide gel electrophoresis (SDS-PAGE), Western blotting, and mass spectrometry) can be performed alone or with the label or other described above. It may be used in combination with a detection method.

  The amount of peptide or polypeptide can also preferably be determined as follows: (a) a solid support comprising a ligand for the peptide or polypeptide as specified above, a sample comprising the peptide or polypeptide And (b) measuring the amount of peptide or polypeptide bound to the support. Preferably, the ligand is selected from the group consisting of nucleic acids, peptides, polypeptides, antibodies and aptamers, preferably present in a fixed form on the solid support. Materials for producing solid supports are well known in the art and include, among others, commercially available column materials, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and And / or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, durasite, wells and reaction tray walls, plastic tubes, and the like. The ligand or agent may be bound to many different carriers. Examples of well-known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amylose, natural and modified cellulose, polyacrylamide, agarose and magnetite. The nature of the carrier can be either soluble or insoluble for the purposes of the present invention. Suitable methods for immobilizing / immobilizing the ligand are well known and include, but are not limited to, ionic, hydrophobic, covalent interactions, and the like. The use of a “suspension array” as an array according to the present invention is also contemplated (Nolan 2002, Trends Biotechnol. 20 (1): 9-12). In such suspension arrays, carriers such as microbeads or microspheres are present in suspension. The array consists of different microbeads or microspheres that may be labeled and carry different ligands. Methods for producing such arrays are commonly known (US Pat. No. 5,744,305), for example based on solid phase chemistry and light sensitive protecting groups.

Preferably, the amount of marker as referred to herein is determined by using the assays described in Examples 1 and 2.
The term “amount” as used herein includes the absolute amount of a polypeptide or peptide, the relative amount or concentration of said polypeptide or peptide, as well as any value or parameter that can be correlated or derived therefrom. Such values or parameters include intensity signal values derived from all specific physical or chemical properties obtained from said peptides by direct measurement, such as intensity values of mass spectra or NMR spectra. In addition, all values or parameters obtained by indirect measurements as specified elsewhere in this specification, such as reaction amounts determined from biological reading systems in response to peptides, or specifically bound ligands The intensity signal obtained from is included. It is to be understood that values correlating to the aforementioned quantities or parameters can also be obtained by all standard mathematical operations.

  The term “compare” means herein the amount of a marker as referred to herein, particularly the amount of peptide or polypeptide contained by the sample to be analyzed, elsewhere in this specification. Comparing to the appropriate reference source amount specified. As used herein, comparing refers to comparing corresponding parameters or values, for example, comparing an absolute amount to an absolute reference amount, while comparing a concentration to a reference concentration, or an intensity signal obtained from a test sample. It should be understood when compared to the same type of intensity signal of the reference sample. The comparison referred to in step (b) of the method of the present invention may be performed manually or by a computing device (eg, of the system disclosed herein). For computer-aided comparisons, it is also possible to compare the value of the determined quantity with a value corresponding to an appropriate reference stored in a database by a computer program. The computer program can further evaluate the results of the comparison, i.e. automatically provide the desired evaluation in an appropriate output format. The results may preferably serve as an aid in assessing whether the subject should be subjected to diagnostic assessment based on imaging methods as shown elsewhere herein. For example, the results of the comparison may be provided as raw data (absolute or relative), and in some cases an indicator in the form of a word, phrase, symbol, or number that can be a specific diagnostic / evaluation indicator As may be provided. Accordingly, the reference amount should be selected to allow any of the differences or similarities in the amounts to be compared to perform the assessment or diagnosis disclosed herein.

  The term “reference amount” as used herein refers to subject in either a group of subjects that should be subjected to diagnostic evaluation based on imaging or a group of subjects that should not be subjected to diagnostic evaluation based on imaging. Refers to the amount that can be allocated. Such a reference amount may be a threshold amount that separates these groups from each other. Accordingly, a reference amount of a biomarker as referred to herein, particularly cardiac troponin or FGF-23, should be subjected to a group of subjects that should be subjected to diagnostic assessment based on imaging, or diagnostic assessment based on imaging The amount should allow the subject to be assigned to a subject group that is not. Appropriate threshold amounts for separating the two groups are referred to herein from either the subject or group of subjects that are to be subjected to the assessment, or from the subject or group of subjects that are not to be subject to the assessment. Based on the amount of marker as mentioned, it can be easily calculated by statistical tests mentioned elsewhere in this specification. Preferred reference amounts that can be obtained from the aforementioned subjects or groups of subjects are set forth elsewhere herein.

  A reference amount can also be used to define or establish a threshold amount. The threshold amount preferably allows for inclusion and / or exclusion of whether the subject should be subjected to diagnostic assessment based on imaging. The inclusion and / or exclusion may be provided by a computing device of the system disclosed herein based on the comparison of the calculated “amount” to a reference or threshold. For example, the computing device of the system may provide an indicator in the form of words, symbols, or numbers that are indicators of inclusion or exclusion. The reference amount applicable to an individual subject can vary depending on various physiological parameters, such as age, gender, or subpopulation, and the means used to determine a polypeptide or peptide referred to herein. . An appropriate reference amount can also be determined from the reference sample to be analyzed together with the test sample, ie simultaneously or subsequently.

  Preferably, the reference amount is a calculated reference amount. Preferably, the calculated reference amount makes it possible to distinguish between subjects that should be subjected to diagnostic evaluation based on imaging and subjects that should not be subjected to diagnostic evaluation based on imaging. To do. The reference amount can in principle be calculated for a cohort of subjects as specified above based on the mean value for a given biomarker by applying standard statistical methods. In particular, the accuracy of a test, for example a method intended to diagnose an event or not, is best described by the receiver operating characteristics (ROC) (in particular Zweig 1993, Clin. Chem. 39: 561-577). See). The ROC graph is a plot of all sensitivity / specificity pairs that result from continuously changing the decision threshold over the entire range of observed data. The clinical performance of a diagnostic method depends on its accuracy, that is, the ability to correctly assign a subject to a particular assessment, prognosis or diagnosis. The ROC plot shows the overlap between the two distributions by plotting sensitivity against 1-specificity over the complete range of thresholds suitable for making a distinction. On the y-axis is the sensitivity or true positive fraction, which is defined as the ratio of the number of true positive test results to the product of the number of true positives and the number of false negative test results . This has also been referred to as positive in the presence of a disease or condition. This is calculated exclusively from the affected subgroup. On the x-axis is the false positive fraction, or 1-specificity, defined as the ratio of the number of false positive results to the product of the number of true negatives and the number of false positive results. This is an indicator of specificity and is calculated entirely from the unaffected subgroup. Since true positive and false positive fractions are calculated completely separately, by using test results from two different subgroups, the ROC plot is independent of the prevalence of events in the cohort. Each point on the ROC plot corresponds to a sensitivity / specificity pair that corresponds to a particular decision threshold. Tests with complete discrimination (no overlap between the two distributions of results) have ROC plots through the upper left corner, where the true positive fraction is 1.0, or 100% (full sensitivity) ) And the false positive fraction is 0 (full specificity). The theoretical plot of the test without discrimination (identical distribution of results for the two groups) is a 45 ° diagonal line from the lower left corner to the upper right corner. Most plots fall between these two extremes. If the ROC plot is completely below the 45 ° diagonal, this is easily corrected by reversing the criterion for “positive” from “greater than” to “less than” or vice versa . Qualitatively, the closer the plot is to the upper left corner, the higher the overall accuracy of the test. Depending on the desired confidence interval, a threshold can be obtained from the ROC curve to allow diagnosis or prediction of a given event with an appropriate balance of sensitivity and specificity, respectively. Thus, the reference value to be used in the above-described method of the present invention can preferably be a threshold value or a cut-off amount, and this is preferably established as an ROC for the cohort as described above. And by obtaining a threshold amount therefrom. Depending on the desired sensitivity and specificity of the diagnostic method, the ROC plot allows to obtain an appropriate threshold.

For cardiac troponin, especially troponin T, the preferred reference amount is in the range of 5-6 pg / ml. A particularly preferred reference amount is 5.9 pg / ml.
For FGF-23, the preferred reference amount is in the range of 73-77 pg / ml. A particularly preferred reference amount is 74 RU / ml. Preferably, the amount of marker is determined by using the assays described in Examples 1 and 2.

Preferably, the following applies as a diagnostic algorithm (especially when the reference is a calculated reference quantity):
Preferably, the increased amount of marker (and thus cardiac troponin and / or FGF-23) in the sample from the subject as compared to the reference amount should subject the subject to imaging-based diagnostic evaluation. It shows that there is. Again preferably, a reduced amount of the marker (s) in the sample from the subject when compared to the reference amount indicates that the subject should not be subjected to diagnostic assessment based on imaging.

  As indicated herein above, the reference amount may be obtained from a subject or group of subjects known to be sensitive to diagnostic assessment based on imaging methods. In this case, the amount of marker (s) to be determined in the context of the present invention that is essentially the same as or higher than the reference amount (s) is determined by imaging the subject to be tested. Indicates that it should be used for diagnostic evaluation based on the law. Preferably, the reference amount is obtained from a sample of the subject or from a sample of the subject group. Preferably, a subject known to be sensitive to diagnostic assessment based on imaging is a subject suffering from abnormal wall fractionation (the term “abnormal medial wall rate” Specified elsewhere in the description).

Preferably, it is possible to include that the subject should be subjected to an imaging-based assessment by applying the aforementioned reference amount.
Additionally or alternatively, the reference amount may preferably be obtained from a subject or group of subjects known not to be sensitive to diagnostic assessment based on imaging. In this case, the amount of marker (s) to be determined in the context of the present invention that is essentially the same as or lower than the reference amount (s) is determined by imaging the subject to be tested. Indicates that it should not be used for legal diagnostic evaluation. Preferably, the reference amount is obtained from a sample of the subject or from a sample of the subject group. Preferably, a subject known not to be sensitive to diagnostic assessment based on imaging is a subject not suffering from an abnormal medial wall rate (see the term “abnormal medial wall rate” herein). To be specified elsewhere).

Preferably, by applying the aforementioned reference amount, it is possible to exclude that the subject should not be subjected to imaging based evaluation.
Furthermore, when the reference amount is obtained from a subject or group of subjects known not to be sensitive to diagnostic assessment based on imaging, particularly from a subject or group of subjects not suffering from abnormal medialization The following applies as diagnostic algorithms: Preferably, the amount of cardiac troponin, in particular troponin T, is at least 10%, or more preferably at least 25% higher than the reference amount of cardiac troponin, in particular troponin T. A measure of the subject that should be subjected to the evaluation. Again preferably, an amount of FGF-23 that is at least 5%, or more preferably at least 10%, or even more preferably at least 25% higher than the reference amount of FGF-23 is subjected to diagnostic assessment based on imaging. An indication of the subject that should be.

  The definition of the term “subject” is provided elsewhere herein. The definition also applies to reference subject (s) according to the methods of the invention (ie, a subject that obtains a reference amount). Preferably, the subject to be tested and the reference subject (s) are of the same age, sex and / or race. Thus, the reference amount is preferably adjusted for age, gender and / or race. Again preferably, the subject to be tested and the reference subject (s) have hypertension, particularly hypertension associated with the same risk factors (see elsewhere in this specification for risk factors). It is also possible to suffer.

  Markers as referred to herein may be determined alone. However, they may also be determined together, i.e. the method of the invention may comprise a determination of cardiac troponin and FGF-23.

  Furthermore, in an embodiment of the invention, in addition to the determination of FGF-23, in step a) the amount of vitamin D is further determined and the ratio between the amount of FGF-23 and the amount of vitamin D is further determined in step a1. ) And the ratio thus calculated is intended to be compared with a reference ratio.

Thus, the present invention is also a method for assessing whether a subject should be subjected to an imaging-based diagnostic assessment comprising:
a) In a sample from the subject, determining the amount of fibroblast growth factor 23 (FGF-23) and the amount of vitamin D;
a1) Calculate the ratio between the amount of FGF-23 and the amount of vitamin D, and b) compare the ratio thus calculated to the reference ratio, thereby subjecting the subject to imaging-based diagnostic evaluation It also relates to said method comprising the step of evaluating whether it should.

The amount of FGF-23 and vitamin D may be determined in different samples from the subject. However, it is also contemplated to determine the amount in the same sample.
Preferably, the method described above can further comprise a determination of the amount of cardiac troponin in the sample (in step a)) and a comparison of the amount thus determined with a reference amount (in step b).

Preferably, the assessment of whether the subject should be subjected to a diagnostic assessment based on imaging is based on the result of the comparison (s) performed in step b).
The ratio between the amount of FGF-23 and the amount of vitamin D may be the ratio of the amount of FGF-23 to the amount of vitamin D, or the ratio of the amount of vitamin D to the amount of FGF-23. May be.

Appropriate reference ratios can be readily determined by those skilled in the art, as indicated above in the context of reference amounts.
Preferably, where the ratio is the ratio of the amount of FGF-23 to the amount of vitamin D, the following is also true as a diagnostic algorithm:
Preferably, an increase in the ratio of the amount of FGF-23 to the amount of vitamin D in the sample derived from the subject as compared to a reference ratio (of the amount of FGF-23 to the amount of vitamin D) images the subject. Indicates that it should be used for diagnostic evaluation based on the law. Again preferably, a decrease in the ratio of the amount of FGF-23 to the amount of vitamin D in the sample from the subject as compared to a reference ratio (of the amount of FGF-23 to the amount of vitamin D) Indicates that it should not be used for diagnostic evaluation based on imaging methods.

  The reference ratio of FGF-23 to vitamin D may be obtained from a subject or group of subjects known to be sensitive to diagnostic assessment based on imaging. In this case, a ratio of FGF-23 to vitamin D that is essentially the same as or higher than the reference ratio indicates that the subject should be subjected to the assessment. Preferably, the reference ratio is obtained from a sample of the subject or from a sample of the subject group. Preferably, the subject known to be subjected to diagnostic assessment based on imaging is a subject suffering from abnormal MFS.

Preferably, it is possible to include that the subject should be subjected to the assessment by applying the reference ratio described above.
Additionally or alternatively, the reference ratio of FGF-23 to vitamin D may preferably be obtained from a subject or group of subjects known not to be sensitive to imaging based diagnostic assessment. In this case, a ratio of FGF-23 to vitamin D in the test sample that is essentially the same as or lower than the reference ratio indicates that the subject should not be subjected to the assessment. Preferably, the reference ratio is obtained from a sample of the subject or from a sample of the subject group. Preferably, a subject known not to be subjected to diagnostic assessment based on imaging is a subject not suffering from abnormal MFS.

Preferably, subjecting the subject to the assessment can be eliminated by applying the reference ratio described above.
Again preferably, if the reference ratio of FGF-23 to vitamin D is obtained from a subject or group of subjects not suffering from abnormal medialization, the following also applies as a diagnostic algorithm: A ratio of FGF-23 to vitamin D in the test sample that is at least 5%, or more preferably at least 10% higher, is an indicator of the subject that should be subjected to imaging based diagnostic assessment.

  The term “vitamin D” is well understood by those skilled in the art. In the present specification, the term is preferably a group of fat-soluble prohormones, the two main types of which are vitamin D2 (also referred to as ergocalciferol) and vitamin D3 (also referred to as cholecalciferol). Is related to said group. Preferably, the term includes any type of vitamin D, in particular vitamin D1, vitamin D2, vitamin D3 or vitamin D4. Furthermore, the term is assumed to include any precursor. Vitamin D is typically obtained in organisms from sun exposure, food, and nutritional supplements, is biologically inactive, and is activated in organisms via two hydroxylation reactions. For example, the active form of vitamin D found in humans is calcitriol.

  It is known in the art that the most accurate method for determining the amount of vitamin D is through the determination of 25-hydroxyvitamin D. 25-hydroxyvitamin D is a prehormone produced in the liver by the hydroxylation of vitamin D3 (cholecalciferol) by the enzyme cholecalciferol 25-hydroxylase. Thus, the term “vitamin D” refers specifically to 25-hydroxyvitamin D. Therefore, in the context of the present invention, it is particularly preferred to determine the amount of 25-hydroxyvitamin D in a sample from the subject.

  In a preferred embodiment of the foregoing method, the reference amount (ratio) of the marker (s) to be determined in the context of the method is obtained before the sample as shown in step a) (test) test The amount (ratio) of marker (s) in a body-derived sample.

  In the context of the present application, the sample obtained before the sample as shown in step a) is also referred to as “first sample”, and the test sample shown in step a) is after the first sample. Is called “second sample”.

Accordingly, the present invention is a method for assessing whether a subject should be subjected to diagnostic assessment based on imaging, comprising:
a) in the first and second samples from the subject, the amount (s) of cardiac troponin and / or fibroblast growth factor 23 (FGF-23) is determined, and b) thus determined Comparing the amount (s) in the second sample with the amount (s) in the first sample, thereby assessing whether the subject should be subjected to imaging-based diagnostic evaluation The method.

  As indicated above, the second sample should have been obtained after the first sample (in other words: the first sample should have been obtained before the second sample). It is specifically envisaged that the second sample is obtained after a reasonable period of time after obtaining the first sample. It is to be understood that the amount of biomarker referred to herein does not change instantaneously (eg, within 1 minute or 1 hour). Thus, “reasonable” in this context refers to the interval between obtaining the first and second samples that allows the biomarker (s) to be adjusted. Preferably, the second sample is obtained 1 to 24 months after the first sample. More preferably, the second sample is obtained 6 to 18 months after the first sample. Even more preferably, the second sample is obtained 6 to 12 months after the first sample. Most preferably, the second sample is obtained 9 to 12 months after the first sample. Thus, the first sample is preferably from 1 to 24 months before, more preferably from 6 to 18 months, even more preferably from 6 to 12 months, or most preferably from 9 to 12 months before the second sample. It is assumed that

  The time course of the amount of marker, ie cardiac troponin and / or FGF-23, as mentioned in the method of the invention (or the ratio between FGF-23 and vitamin D in a subject as indicated herein) It is also envisaged to evaluate (elapsed time). Thus, the method described above comprises the amount of cardiac troponin and / or FGF-23 in at least one additional sample from the subject (and thus in the third sample, in the fourth sample, in the fifth sample, etc.). (Or the ratio between FGF-23 and vitamin D) and the amount thus determined (ratio) is determined from said first sample and / or said second sample and / or said at least one It may further comprise a further step of comparing the ratio of the amount of cardiac troponin and FGF-23 (or the ratio between FGF-23 and vitamin D) in any sample obtained before obtaining further samples, respectively. Is possible. See above for preferred time intervals for obtaining samples.

  Preferably, an increase in the amount of cardiac troponin and / or FGF-23 (and / or the ratio of the amount of FGF-23 to the amount of vitamin D) in the second sample as compared to the first sample, and more A significant increase, and most preferably a statistically significant increase, indicates that the subject should be subjected to diagnostic assessment based on imaging. Preferably, a reduction in the amount of cardiac troponin and / or FGF-23 (and / or the ratio of the amount of FGF-23 to the amount of vitamin D) in the second sample as compared to the first sample, and more Preferably a significant reduction, and most preferably a statistically significant reduction, or an essentially unchanged amount (or an essentially unchanged ratio) of cardiac troponin and / or FGF-23, subject the subject to imaging. Indicates that it should not be subjected to a diagnostic assessment based on

In particular, a significant increase is an increase in size that is considered significant with respect to the assessment, in particular said increase is considered statistically significant.
The terms “significant” and “statistically significant” are known to those skilled in the art. Thus, whether an increase or decrease is significant or statistically significant can be readily determined by those skilled in the art using a variety of well-known statistical evaluation tools.

  During the course of the present invention, the amount of cardiac troponin and / or FGF-23 (or vitamin D) that has been found as an indicator of subjects that should or should not be subjected to diagnostic assessment based on imaging A significant increase in the amount (ratio) of biomarker (s) in a blood, serum or plasma sample, preferably in the blood, serum or plasma sample, is provided herein below.

If the marker is cardiac troponin, the following applies:
Preferably, the amount of cardiac troponin, especially troponin T, in the second sample that is at least 10%, or more preferably at least 25% higher than the reference amount (the amount of cardiac troponin in the first sample) is based on imaging An index of a subject to be subjected to a diagnostic evaluation.

  Further, according to the present invention, preferably at least 0.5 pg / ml, more preferably at least 1.0 pg / ml, and even more preferably at least 1.5 pg / ml, at least 2.0 pg / ml, or at least 2.5 pg. An increase in the amount of cardiac troponin, particularly troponin T, in the second sample compared to the amount in the first sample, at least 3.0 pg / ml, and most preferably at least 4.0 pg / ml is significant Indicating that the subject should be subjected to imaging-based diagnostic evaluation.

When the marker is FGF-23, the following is true:
Preferably, the amount of FGF-23 in the second sample that is at least 5%, or more preferably at least 10% higher than the reference amount (the amount of FGF-23 in the first sample) is a diagnostic assessment based on imaging. It is an index of the subject to be subjected to.

  Further, according to the present invention, preferably at least 1.0 pg / ml, more preferably at least 2.0 pg / ml, and even more preferably at least 3.0 pg / ml, at least 4.0 pg / ml, or at least 5.0 pg. An increase in the amount of FGF-23 in the second sample as compared to the amount in the first sample, at least 6.0 pg / ml, and most preferably at least 7.0 pg / ml, is significant. And thus indicates that the subject should be subjected to diagnostic assessment based on imaging.

When determining the ratio of FGF-23 to vitamin D, the following applies:
Preferably, the ratio in the second sample that is at least 5%, or more preferably at least 10% higher than the reference ratio (ratio in the first sample) is the subject's subject to be subjected to imaging based diagnostic assessment. It is an indicator.

  In a preferred embodiment of the foregoing method, the subject to be tested does not suffer from abnormal MFS when the first sample is obtained. Furthermore, it is preferred that the subject does not suffer from diastolic dysfunction and / or systolic dysfunction when the first sample is obtained. However, the subject is suffering from hypertension when the first sample is obtained. Hypertension may be accompanied by risk factors as shown elsewhere in this specification. Preferably, this is classified as a Stage A subject according to the ACC / AHA classification as mentioned above when the first sample is obtained.

Method for Diagnosing Abnormal Middle Wall Shortening Rate The definitions and explanations provided herein above apply mutatis mutandis to the following.

The present invention also provides a method for diagnosing an abnormal medial wall shortening rate (MFS) in a subject comprising:
a) in the sample from the subject, the amount of cardiac troponin and / or fibroblast growth factor 23 (FGF-23) is determined, and b) the amount (s) thus determined is replaced by a reference amount (single or The method also comprises the step of diagnosing an abnormal medial wall shortening rate as compared to the plurality.

  Preferably, c) whether the subject suffers from abnormal MFS by performing further steps, assessing whether the subject suffers from abnormal MFS based on the results of the comparison performed in step b) evaluate.

  The method of the present invention is preferably an ex vivo or in vitro method. Furthermore, steps may be included in addition to those explicitly mentioned above. For example, further steps may relate to sample pretreatment or evaluation of results obtained by the method. The method may be performed manually or may be assisted by automation. Preferably, steps (a) and / or (b) are wholly or partially for computer-implemented comparison and / or distinction based on the determination in step (a) or the comparison in step (b), It may be assisted by automation, such as a suitable robot and sensor device.

  The term “diagnosing” as used herein preferably means assessing whether a subject as referred to herein suffers from an abnormal medial wall shortening rate (MFS). The term “abnormal middle wall shortening rate” is defined elsewhere in this specification.

  As will be appreciated by those skilled in the art, such an assessment is usually not intended to be correct for 100% of the subjects to be diagnosed. However, the term requires that the assessment is correct for a statistically significant portion of the subject (eg, a cohort in a cohort study). Whether a part is statistically significant is easily determined by those skilled in the art using a variety of well-known statistical evaluation tools such as confidence interval determination, p-value determination, Student's t-test, Mann-Whitney test, etc. Is possible. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001.

  The term “reference amount” is defined elsewhere in this specification. In the context of the foregoing method, the term refers to subject to either a group of subjects suffering from abnormal MFS or a group of subjects not suffering from abnormal MFS (and thus having normal MFS). Refers to the amount that can be allocated. Such a reference amount may be a threshold amount that separates these groups from each other. Thus, a reference amount of cardiac troponin or FGF-23, particularly for a biomarker as referred to herein, can be applied to a group of subjects suffering from abnormal MFS or a group of subjects not suffering from abnormal MFS. The amount should allow the subject to be assigned. An appropriate threshold amount to separate the two groups is either a subject or group of subjects known to suffer from abnormal MFS, or a subject or group of subjects known to suffer from abnormal MFS. Based on the amount of marker as mentioned herein, from which it can be easily calculated by a statistical test mentioned elsewhere in this specification. Preferred reference amounts that can be obtained from the aforementioned subjects or groups of subjects are set forth elsewhere herein.

Preferably, the following applies as a diagnostic algorithm:
Preferably, the increased amount (s) of biomarker in the sample from the subject as compared to the reference amount (s) indicates that the subject suffers from abnormal MFS and the reference amount A reduced amount (s) in a sample from the subject when compared to the singular (s) indicates that the subject does not suffer from abnormal MFS. Preferably, the subject not suffering from abnormal MFS has normal MFS.

  As indicated herein above, the reference amount can be derived from a subject or group of subjects known to suffer from abnormal MFS. In this case, the amount (s) of the marker (s) determined in step a) that is essentially the same as or higher than the reference amount (s) is to be tested. Indicates that the subject suffers from abnormal MFS. Preferably, the reference amount is obtained from a sample of the subject or from a sample of the subject group.

Preferably, abnormal MFS can also be included by applying the aforementioned reference amount.
Additionally or alternatively, the reference amount can preferably be derived from a subject or group of subjects known not to suffer from abnormal MFS. In this case, the amount (s) of the marker (s) determined in step a) that is lower than the reference amount (s) indicates that the subject does not suffer from abnormal MFS. Preferably, the reference amount is obtained from a sample of the subject or from a sample of the subject group.

Preferably, it is possible to eliminate abnormal MFS by applying the aforementioned reference amount.
Preferably, if the reference amount (s) is obtained from a subject or group of subjects known not to suffer from abnormal medialization, the following may also apply as a diagnostic algorithm: An amount of cardiac troponin, particularly troponin T, that is at least 10%, or more preferably at least 25% higher than a reference amount of cardiac troponin, particularly troponin T, is an indicator of a subject suffering from abnormal MFS. Again, preferably the amount of FGF-23 that is at least 5%, or more preferably at least 10% higher than the reference amount of FGF-23 is an indication of a subject suffering from abnormal MFS.

  The definition of the term “subject” is provided elsewhere herein. The definition also applies to the reference subject (s). Preferably, the subject to be tested and the reference subject (s) have approximately the same age (+/− year, preferably the same age), the same gender and / or race. Again preferably, the subject to be tested and the reference subject (s) have hypertension, particularly hypertension associated with the same risk factors (see elsewhere in this specification for risk factors). It is also possible to suffer.

  Furthermore, in an embodiment of the invention, in addition to the determination of FGF-23, in step a) the amount of vitamin D is further determined, and in a further step a1) between the amount of FGF-23 and the amount of vitamin D It is intended to calculate the ratio of and to compare the ratio thus calculated with the reference ratio.

Accordingly, the present invention is also a method for diagnosing abnormal medial wall shortening rate (MFS) in a subject comprising the steps of:
a) In a sample from the subject, determining the amount of fibroblast growth factor 23 (FGF-23) and the amount of vitamin D;
a1) calculating the ratio between the amount of FGF-23 and the amount of vitamin D; and b) comparing the ratio thus calculated to the reference ratio, thereby diagnosing an abnormal medial wall shortening rate. It also relates to said method comprising.

The amount of FGF-23 and vitamin D may be determined in different samples from the subject. However, it is specifically contemplated to determine the amount in the same sample.
Optionally, the method can further comprise determining the amount of cardiac troponin in the sample (in step a) and comparing the amount thus determined to the reference amount (in step b).

Preferably, the assessment of whether the subject should be subjected to a diagnostic assessment based on imaging is based on the result of the comparison (s) performed in step b).
The ratio between the amount of FGF-23 and the amount of vitamin D may be the ratio of the amount of FGF-23 to the amount of vitamin D, or the ratio of the amount of vitamin D to the amount of FGF-23. May be.

Appropriate reference ratios can be readily determined by those skilled in the art, as indicated above in the context of reference amounts.
Preferably, where the ratio is the ratio of the amount of FGF-23 to the amount of vitamin D, the following applies as a diagnostic algorithm:
Preferably, the increase in the ratio of the amount of FGF-23 to the amount of vitamin D in the sample from the subject, as compared to the reference ratio (of the amount of FGF-23 to the amount of vitamin D), indicates that the subject has MFS Show that you suffer from. Again preferably, the decrease in the ratio of the amount of FGF-23 to the amount of vitamin D in the sample from the subject when compared to the reference ratio (of the amount of FGF-23 to the amount of vitamin D) is Indicates that the patient does not suffer from abnormal MFS.

Preferably, when determining the ratio of FGF-23 to vitamin D, the following is also true:
The reference ratio of FGF-23 to vitamin D may be obtained from a subject or group of subjects known to suffer from abnormal MFS. In this case, a ratio of FGF-23 to vitamin D that is essentially the same as or higher than the reference ratio indicates that the subject to be tested suffers from abnormal MFS. Preferably, the reference ratio is obtained from a sample of the subject or from a sample of the subject group.

  Additionally or alternatively, a reference ratio of FGF-23 to vitamin D may be obtained from a subject or group of subjects known not to suffer from abnormal MFS. In this case, a ratio of FGF-23 to vitamin D in the test sample that is essentially the same as or lower than the reference ratio indicates that the subject does not suffer from abnormal MFS. Preferably, the reference amount is obtained from a sample of the subject or from a sample of the subject group.

  Preferably, when the reference ratio of FGF-23 to vitamin D is obtained from a subject or group of subjects known not to suffer from abnormal medialization, the following also applies as a diagnostic algorithm: The ratio of FGF-23 to vitamin D in the test sample, which is at least 5%, or more preferably at least 10% higher than the reference ratio, is an indication of a subject suffering from abnormal MFS.

  In particular, a preferred reference ratio obtained from a subject or group of subjects known not to suffer from an abnormal medialization is the median ratio in such a group of subjects. Preferably, the reference ratio is 5.3.

  In a preferred embodiment, the method of the invention is used when the subject to be tested suffers from abnormal MFS and / or when the subject to be tested is to be subjected to imaging-based diagnostic evaluation Further comprising the step of recommending therapy.

Preferred therapies as used in the context of the present invention include lifestyle changes, dietary treatments, interventions on the body, and administration of appropriate agents for the treatment of subjects.
Suitable drugs for treatment are well known in the art, see for example Heart Disease, 2008, 8th edition, Braunwald, Elsevier Sounders, Chapter 24 (for heart failure) and Chapter 41 (for hypertension) I want to be. These treatments are part of the present invention.

Lifestyle changes include smoking cessation, appropriate alcohol consumption, increased physical activity, weight loss, sodium (salt) restriction, weight management and a healthy diet, daily fish oil, and salt restriction.
The therapy may also include intervention. One preferred intervention in the context of the present invention is the administration of an antihypertensive drug, an angiotensin converting enzyme (ACE) inhibitor, an angiotensin II receptor blocker (ARB), an aldosterone antagonist, a beta blocker for diuretics, and calcium. Antagonists are preferred.

Preferably, administration of one or more of the following drugs should be recommended:
Diuretics such as loop diuretics, thiazides and thiazide-like diuretics, potassium-sparing diuretics, type I mineralocorticoid receptor antagonists, antialdosterone, carbonic dehydrogenase inhibitors, vasopressure antagonists.

Beta blockers such as proprenolol, metoprolol, bisoprolol, carvedilol, bucindolol, nebivolol; calcium antagonists such as dihydropyridine, verapamil, diltiazem;
Adrenergic agents such as dobutamine, dopamine, epinephrine, isoprotenerol, norepinephrine, phenylephrine;
Positive inotropic agents such as digoxin, digitoxin;
ACE inhibitors such as enalapril, captopril, ramipril, trandolapril;
Angiotensin receptor antagonists such as losartan, valsartan, irbesartan, candesartan, telmisartan, eprosartan;
Adsterone antagonists such as epleron, spironolactone, canrenone, mexrenone, prorenone: statins, especially atorvastatin, fluvastatin, lovastatin, pravastatin, rosuvastatin, simvastatin;
Hydazarin and isosorbide dinitrate.

Method for predicting the risk of death and / or cardiovascular event The present invention is a method for predicting the risk of death and / or cardiovascular event in a subject comprising:
a) in the sample from said subject, the amount of IGFBP7 and / or the amount of FGF-23 is determined, and b) the amount (s) as determined in step a) is changed to the reference amount (s) ), Thereby further predicting the risk of death and / or cardiovascular events in the subject.

  In the foregoing method embodiment, in addition to the determination of FGF-23, in step a), the amount of vitamin D is further determined to calculate the ratio between the amount of FGF-23 and the amount of vitamin D; And it is intended to compare the ratio thus calculated with the reference ratio.

Accordingly, the present invention is also a method for predicting the risk of death and / or cardiovascular events in a subject comprising:
a) In a sample from the subject, determining the amount of fibroblast growth factor 23 (FGF-23) and the amount of vitamin D;
a1) Calculate the ratio between the amount of FGF-23 and the amount of vitamin D, and b) compare the ratio thus calculated to the reference ratio, thereby predicting the risk of death and / or cardiovascular events Including the above-described method.

Of course, in addition to the amounts of FGF-23 and vitamin D, the amount of IGFBP7 may also be determined.
Accordingly, the present invention is also a method for predicting the risk of death and / or cardiovascular events in a subject comprising:
a) determining the amount of IGFBP7, fibroblast growth factor 23 (FGF-23) and vitamin D in a sample from the subject;
a1) Calculate the ratio between the amount of FGF-23 and the amount of vitamin D;
b) comparing the ratio thus calculated with the reference ratio, and b1) comparing the amount of IGFBP7 to the reference ratio,
It also relates to the method, comprising predicting the risk of death and / or cardiovascular events in the subject.

  The ratio between the amount of FGF-23 and the amount of vitamin D may be the ratio of the amount of FGF-23 to the amount of vitamin D, or the ratio of the amount of vitamin D to the amount of FGF-23. May be.

  Preferably, c) death and / or by performing further steps that predict the risk of death and / or cardiovascular events based on the results of the comparison performed in step b) (or steps b) and b1)) Predict the risk of cardiovascular events.

The amount of biomarker may be determined in different samples from the subject. However, it is also contemplated to determine the amount in the same sample.
In a preferred embodiment, the method for predicting the risk of mortality and / or cardiovascular events comprises in a sample from a subject an amount of brain natriuretic peptide, in particular BNP or NT-proBNP, and / or cardiac troponin, in particular Further comprising determining the amount of troponin T or I and comparing the amount of said brain natriuretic peptide and / or the amount of cardiac troponin to a reference amount or amounts.

  The method of the present invention is preferably an ex vivo or in vitro method. Further, the method may include steps in addition to those explicitly mentioned above. For example, further steps may relate to sample pretreatment or evaluation of results obtained by the method. The method of the present invention can also be used for monitoring, validation and subclassification. The method may be performed manually and / or the method may be assisted by automation. Preferably, steps (a), (b) and / or (c) are wholly or partly automated, eg for determination in step (a) or in a computer-implemented comparison in step (b) May be assisted by a suitable robot and sensor device for

  The term “predicting” as used herein refers to the death of a subject (eg, death caused by heart failure) and / or cardiovascular events, preferably acute, within a future defined time window (prediction window). Refers to assessing the probability of developing a cardiovascular event, such as acute coronary syndrome (ACS). The prediction window is the interval at which a subject will develop a cardiovascular event or die according to the predicted probability. The prediction window can be the life expectancy of the subject upon analysis by the method of the present invention. Preferably, however, the prediction window is 1 year, 2 years, 3 years after performing the method of the present invention (more preferably and more accurately after obtaining a sample to be analyzed by the method of the present invention). 4 years, 5 years, 10 years, 15 years or 20 years. Most preferably, the prediction window is an interval of 4 years or 5 years. As will be appreciated by those skilled in the art, such an assessment is usually not intended to be correct for 100% of the subjects to be analyzed. The term, however, requires that the assessment be valid for a statistically significant portion of the subject to be analyzed. Whether a part is statistically significant can be easily determined by those skilled in the art using a variety of well-known statistical evaluation tools such as confidence interval determination, p-value determination, Student's t-test, Mann-Whitney test, etc. It is. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001. Preferably, the probability envisaged by the present invention allows the prediction to be correct for at least 60%, at least 70%, at least 80%, or at least 90% of subjects in a given cohort.

  The term “death” as used herein preferably relates to death from any cause, and more preferably death from a cardiovascular event. The term “cardiovascular event” as used herein refers to any disorder of the cardiovascular system, and preferably includes any acute cardiovascular event. The acute cardiovascular event is preferably stable angina (SAP) or acute coronary syndrome (ACS). ACS patients can also exhibit unstable angina (UAP) or myocardial infarction (MI). The MI can also be ST-elevated MI (STEMI) or non-ST-elevated MI (NSTEMI). NSTE-ACS as used herein includes UAP and NSTEMI. The onset of MI can be followed by left ventricular dysfunction (LVD), development of heart failure, or even death. Further preferred cardiovascular events include cardiac bradyarrhythmias or tachyarrhythmias, including sudden cardiac death and stroke (cerebrovascular events or accidents). Death can also refer to mortality or the ratio of deaths to a given population of subjects.

  The expression “predicting the risk of death and / or cardiovascular events” as used herein refers to reducing the subject to be analyzed by the method of the present invention to a group of subjects having an increased risk or Is assigned to one of the groups with the given risk. The elevated risk, as referred to in accordance with the present invention, preferably develops a cardiovascular event for the subject with respect to the average risk of cardiovascular event or cardiac death in the subject population within a predetermined prediction window. Means that the risk or risk of death is significantly increased (ie, significantly increased). The reduced risk as referred to in accordance with the present invention is preferably the risk of developing a cardiovascular event for a subject with respect to the average risk of cardiovascular event or cardiac death in a subject population within a predetermined prediction window. Or it means that the risk of death is significantly reduced. In particular, a significant increase or decrease in risk is an increase or decrease in size risk that is considered significant with respect to prognosis, in particular, said increase or decrease is considered statistically significant. The terms “significant” and “statistically significant” are known to those skilled in the art. Thus, whether an increase or decrease in risk is significant or statistically significant can be readily determined by those skilled in the art using a variety of well-known statistical assessment tools.

  Preferably, for a 4 year (or 5 year) prediction window, the increased risk of death (or cardiovascular event) is in the range of 8.0% to 19.0%, more preferably 12.0% to 17 Within the range of 0.0%, most preferably within the range of 8.0% to 16.0%. The increased and thus increased risk of mortality herein is preferably higher than 8.0%, preferably higher than 12.0%, preferably for a 4 year (or 5 year) prediction window More preferably higher than 17%, even more preferably higher than 20%. As used herein, the reduced risk of death (or cardiovascular event) is preferably less than 8.0%, preferably less than 6%, even more preferably less than 4%, with respect to a 4 year prediction window, And most preferably, it is in the range of 3.0% to 8.0%.

  The term “subject” is as defined above. The definition of the term “subject” also applies to the methods described above. Thus, in connection with a method for assessing whether a subject should be subjected to imaging-based diagnostic assessment, as indicated herein above, the subject has, for example, a normal ventricular weight. And

  In the context of the foregoing methods of the invention for predicting the risk of death and / or cardiovascular events, the subject preferably does not suffer from heart failure and / or does not show obvious symptoms of heart failure (as described above) See). Thus, the subject shall not suffer from heart failure classified as stage C or D according to the ACC / AHA classification (see reference above). Further, as will be shown herein below, the patient does not have a reduced left ventricular ejection fraction.

  In a preferred embodiment of the foregoing method, the subject preferably suffers from heart failure classified as stage A or stage B according to the ACC / AHA classification. In another preferred embodiment of the foregoing method, the subject is healthy with respect to heart disease and disorders. A subject that is healthy with respect to heart disease and disorder is considered a subject who does not suffer from heart disease and disorder.

  Preferably, in the context of the method described above, the subject does not suffer from systolic dysfunction (particularly systolic dysfunction with reduced ejection fraction) and / or systolic dysfunction. Preferably, the subject has a retained left ventricular ejection fraction. Thus, the subject to be tested can preferably have a left ventricular ejection fraction (LVEF) greater than 55%, more preferably greater than 60%, and most preferably greater than 65%. .

  The insulin-like growth factor binding protein (IGFBP) system plays an important role in cell proliferation and differentiation. The protein includes two ligands, IGF-I and IGF-II, two receptor type 1 and type 2 IGF receptors, and as of 1995, six IGF binding proteins (IGFBP), IGFBP-1 to -6. (Jones, JI et al., Endocr. Rev. 16 (1995) 3-34). Recently, the IGFBP family has been expanded to include IGFBP-related protein (IGFBP-rP), which has significant structural similarity to IGFBP (Hwa, V. et al., Endocr. Rev 20 (1999) 761- 787). Thus, the IGFBP superfamily not only shares six conventional IGFBPs with high affinity for IGF and the conserved amino-terminal domain of IGFBP, but also exhibits some degree of affinity for IGF and insulin At least 10 IGFBP-rP. IGFBP-rP is a group of cysteine-rich proteins that control a variety of cellular functions, such as cell proliferation, cell adhesion and migration, and extracellular matrix synthesis. In addition, these proteins may be involved in biological processes such as tissue growth and differentiation, regeneration, angiogenesis, wound repair, inflammation, fibrosis, and tumor development (Hwa, V. et al., Endocr Rev 20 (1999) 761-787).

  IGF binding protein 7 (= IGFBP7) is a 30 kDa modular glycoprotein known to be secreted by endothelial cells, vascular smooth muscle cells, fibroblasts, and epithelial cells (Ono, Y. et al., Biochem Biophys Res). Comm 202 (1994) 1490-1496). In the literature, this molecule is also referred to as FSTL2; IBP7; IGF-binding protein-related protein I; IGFBP 7; IGFBP 7v; IGFBP rP1; IGFBP7; IGFBPRP1; It has also been referred to as MAC25; MAC25 protein; PGI2 stimulator; and PSF or prostacyclin stimulator. Northern blot studies revealed wide expression of this gene in human tissues, including heart, brain, placenta, liver, skeletal muscle, and pancreas (Oh, Y. et al., J. Biol. Chem. 271 (1996). ) 30322-30325).

  IGFBP7 was first identified as a gene that is differentially expressed in normal leptomeningeal and mammary epithelial cells compared to its counterpart tumor cells and was named meningioma-related cDNA (MAC25) (Burger) AM et al., Oncogene 16 (1998) 2459-2467). The expressed protein is a tumor-derived adhesion factor (later renamed angiomodulin) (Sprenger, CC et al., Cancer Res 59 (1999) 2370-2375) and prostacyclin stimulating factor (Akagi, K. et al., Proc Natl Acad Sci USA 93 (1996) 8384-8389). Furthermore, the molecule has also been reported as T1 A12, a down-regulated gene in breast cancer (StCroix, B. et al., Science 289 (2000) 1197-1202).

  Preferably, the term “IGFBP7” refers to human IGFBP7. The sequence of the protein is well known in the art and is accessible, for example, through GenBank (NP — 001240764.1). IGFBP7 herein preferably also includes variants of certain IGFBP7 polypeptides. See above for a description of the term “variant”.

As indicated above, the aforementioned methods can further include determining the amount of brain natriuretic peptide and / or cardiac troponin in a sample from the subject. The term “cardiac troponin” has been described elsewhere herein. As used herein, the term "brain natriuretic peptide" preferably refers to a variant thereof having a brain natriuretic (B rain N atriuretic P eptide) (BNP) -type peptide and the same predictive power. BNP-type peptides include preproBNP, proBNP, NT-proBNP, and BNP. The prepro peptide (134 amino acids in the case of prepro BNP) contains a short signal peptide that is enzymatically cleaved to release the pro peptide (108 amino acids in the case of pro BNP). The propeptide is further cleaved into an N-terminal propeptide (NT-propeptide, 76 amino acids for NT-proBNP) and an active hormone (32 amino acids for BNP). Preferably, the brain natriuretic peptide according to the present invention is NT-proBNP, BNP, and variants thereof. BNP is an active hormone and has a shorter half-life than its inactive counterpart NT-proBNP. BNP is metabolized in the blood, while NT-proBNP circulates in the blood as an intact molecule and is excreted in the kidney as such. The in vivo half-life of NT-proBNP is 120 minutes longer than the half-life of BNP, which is 20 minutes (Smith 2000, J Endocrinol. 167: 239-46.). Pre-analysis is more robust with NT-proBNP and can easily transport samples to a central laboratory (Mueller 2004, Clin Chem Lab Med 42: 942-4.). The blood sample may be stored at room temperature for several days, or it can be mailed or transported without recovery loss. In contrast, storing BNP for 48 hours at room temperature or 4 ° C. leads to a concentration loss of at least 20% (Mueller, ibid .; Wu 2004, Clin Chem 50: 867-73.). Thus, depending on the time course or the property of interest, measurement of either the active or inactive form of the natriuretic peptide may be preferred. The most preferred natriuretic peptide according to the present invention is NT-proBNP or a variant thereof. As briefly discussed above, human NT-proBNP, when referred to in accordance with the present invention, is preferably a polypeptide comprising 76 amino acids in length corresponding to the N-terminal portion of the human NT-proBNP molecule. . The structure of human BNP and NT-proBNP has already been described in detail in the prior art, for example WO 02/089657, WO 02/083913 or Bonow, ibid. Preferably, human NT-proBNP is human NT-proBNP, as disclosed herein in EP 0 648 228 B1. These prior art documents are hereby incorporated by reference with respect to the specific sequences of NT-proBNP and variants disclosed therein. The NT-proBNP referred to in accordance with the present invention further includes allelic and other variants of the specific sequence of human NT-proBNP discussed above. Specifically, at the amino acid level, human NT-proBNP, preferably over the entire length of human NT-proBNP, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, Variant polypeptides that are 92%, 95%, 97%, 98%, or 99% identical are envisioned. The degree of identity between two amino acid sequences can be determined by algorithms well known in the art. Preferably, the degree of identity can be determined by comparing two optimally aligned sequences over a comparison window, wherein the fragments of amino acid sequences in the comparison window are optimally aligned. Thus, additions or deletions (eg gaps or overhangs) may be included when compared to a reference sequence (not including additions or deletions). Determine the number of positions where the same amino acid residue is present in both sequences to obtain the number of matched positions, divide the number of matched positions by the total number of positions in the comparison window, and add 100 to the result. Calculate the percentage by multiplying by to get the percent sequence identity. Optimal alignment of sequences for comparison is described by Smith and Waterman Add. APL. Math. 2: 482 (1981), according to the Needleman and Wunsch J. et al. Mol. Biol. 48: 443 (1970) by the homology alignment algorithm of Pearson and Lipman Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988) by the computerized implementation of these algorithms (GAP, BESTFIT, BLAST, PASTA, and TFSTA, Genetics Computer Group (GCG) in Wisconsin Genetics Software Package (75) Science Dr., Madison, Wis.) Or by visual inspection. If two sequences are identified for comparison, preferably GAP and BESTFIT are used to determine the optimal alignment and therefore the degree of identity. Preferably, default values of 5.00 for gap weight and 0.30 for gap weight length are used. The variants referred to above may be allelic variants or any other species-specific homologue, paralog, or ortholog. Proteolytic degradation products still recognized by diagnostic means or by ligands directed against the respective full-length peptides are substantially similar and are also envisioned. Also included are variant polypeptides having amino acid deletions, substitutions, and / or additions compared to the amino acid sequence of human NT-proBNP, so long as the polypeptide has NT-proBNP properties. NT-proBNP properties as referred to herein are immunological and / or biological properties. Preferably, the NT-proBNP variant has immunological properties (ie, epitope composition) comparable to that of human NT-proBNP. Thus, the variant shall be recognizable by the aforementioned means or ligand used for the determination of the amount of natriuretic peptide. Biological and / or immunological NT-proBNP properties are described in Karl et al. (Karl 1999, Scand J Clin Lab Invest 230: 177-181), Yeo et al. (Yeo 2003, Clinica Chimica Acta 338: 107-115). Detectable by the described assay. Variants also include post-translationally modified peptides such as glycosylated peptides. Furthermore, variants according to the invention are also peptides or polypeptides that have been modified after sample collection, for example by covalent or non-covalent attachment of a label to the peptide, in particular a radioactive or fluorescent label.

  The term “reference amount” is as defined above. In the context of the foregoing method, a reference amount of FGF-23, IGFBP7, cardiac troponin, or brain natriuretic peptide, particularly for biomarkers as referred to herein, increases the risk of death and / or cardiovascular events The amount should allow the subject to be assigned to a selected group of subjects or to a group of subjects with reduced risk of death and / or cardiovascular events. A suitable threshold amount that separates the two groups reduces the risk of death and / or cardiovascular events for a subject or group of subjects known to have increased risk of death and / or cardiovascular events Easily calculated from any subject or group of subjects known to be based on the amount of a marker as referred to herein, by a statistical test referred to elsewhere herein Is possible. Preferred reference amounts that can be obtained from the aforementioned subjects or groups of subjects are set forth elsewhere herein.

Preferably, the following applies as a diagnostic algorithm:
Preferably, the reference amount is obtained from a subject or group of subjects known to be at increased risk of death and / or cardiovascular events. In this case, the amount of marker FGF-23 and / or IGFBP7, which is essentially the same as or greater than the reference amount (s), is determined by the death and / or cardiovascular of the subject to be tested. Indicates an increased risk of the event. The same applies in the context of the present invention to markers that may be further determined, namely cardiac troponin and brain natriuretic peptide.

  Additionally or alternatively, the reference amount can be obtained from a subject or group of subjects known to have a reduced risk of death and / or cardiovascular events. In this case, the amount of marker FGF-23 and / or IGFBP7, which is essentially the same as or lower than the reference amount (s), is the mortality and / or cardiovascular volume of the subject to be tested. Indicates that the risk of the event is decreasing. The same applies in the context of the present invention to markers that may be further determined, namely cardiac troponin and brain natriuretic peptide.

  Furthermore, it is assumed that the reference amount is a calculated reference amount. Preferably, the calculated reference amount distinguishes between subjects with an increased risk of death and / or cardiovascular events and subjects with a reduced risk of death and / or cardiovascular events. Shall be possible. Preferably, the increased amount (s) of the marker (s) is an indicator of a subject at increased risk of death and / or cardiovascular events, while the marker (s) Reduced amount (s) is an indication of a subject having a reduced risk of death and / or cardiovascular events.

  As indicated above, the foregoing method may include a combined determination of FGF-23 and vitamin D, and calculation of a reference ratio. Appropriate reference ratios can be readily determined by those skilled in the art, as indicated above, in the context of reference amounts.

  A preferred reference ratio for the ratio of the amount of FGF-23 to the amount of vitamin D is in the range of 4-8.5, in particular in the range of 6.5-8.5. The further reference ratio of the amount of FGF-23 to the amount of vitamin D is preferably 6.5, more preferably 7.5, or most preferably 8.5.

Preferred reference amounts are in the range of 100-115 ng / ml for IGFBP7. A particularly preferred reference amount is 115 ng / ml.
Preferably, where the ratio is the ratio of the amount of FGF-23 to the amount of vitamin D, the following is also true as a diagnostic algorithm:
Preferably, the increase in the ratio of the amount of FGF-23 to the amount of vitamin D in the sample from the subject, as compared to the reference ratio (of the amount of FGF-23 to the amount of vitamin D) is the death of the subject. And / or indicates an increased risk of cardiovascular events. Again preferably, the decrease in the ratio of the amount of FGF-23 to the amount of vitamin D in the sample from the subject as compared to the reference ratio (of the amount of FGF-23 to the amount of vitamin D) is Indicates a reduced risk of death and / or cardiovascular events.

  Again preferably, the reference ratio of FGF-23 to vitamin D can be from a subject or group of subjects known to be at increased risk of death and / or cardiovascular events. In this case, a ratio of FGF-23 to vitamin D that is essentially the same as or greater than the reference ratio indicates that the subject is at increased risk of death and / or cardiovascular events.

  Additionally or alternatively, the reference ratio of FGF-23 to vitamin D can preferably be derived from a subject or group of subjects known to have a reduced risk of death and / or cardiovascular events. . In this case, a ratio of FGF-23 to vitamin D in the test sample that is essentially the same as or lower than the reference ratio reduces the risk of mortality and / or cardiovascular events in the subject. Indicates.

Methods for diagnosing early stage left ventricular hypertrophy (LVH) In the study underlying the present invention, combined determination of endostatin and a BNP-type peptide (eg NT-proBNP and / or BNP), or endostatin and The combined determination of cardiac troponin has been shown to allow reliable identification of subjects with early stage left ventricular hypertrophy. The combined determination of these markers is improved because the determination of endostatin on the one hand and BNP-type peptide or cardiac troponin on the other allows the identification of different subgroups of subjects who are early stage LVH. Cause a diagnosis. An increased amount of one marker or an increased amount of two markers is an indicator of early stage LVH. When determining the amount of only one marker, some patients with early stage LVH will be missed.

  The combined determination of markers has a retained left ventricular ejection fraction, especially to allow an intermediate phenotype that is ongoing from hypertension to contraction or diastolic dysfunction before LVH becomes more prominent Suitable in the subject.

Thus, the present invention is a method for diagnosing early stage left ventricular hypertrophy in a subject with retained left ventricular ejection fraction,
a) determining the amount of endostatin in a sample from the subject, and b) determining the amount of BNP-type peptide and / or cardiac troponin in the sample from the subject, and c) steps a) and b) Comparing the amount as determined in step 1 to a reference amount, thereby diagnosing early stage left ventricular hypertrophy.

  Preferably, the initial stage left ventricular hypertrophy is performed by d) diagnosing whether the subject is in the early stage left ventricular hypertrophy based on the results of the comparison performed in step c). Diagnosed.

The amount of marker in steps a) and b) may be determined in the same sample from the subject or may be determined in different samples from the subject.
The term “diagnosing”, as used herein, preferably means assessing whether a subject as referred to herein in the context of the foregoing method is suffering from early stage LVH. As will be appreciated by those skilled in the art, such an assessment is usually not intended to be correct for 100% of the subjects to be diagnosed. However, the term requires that the assessment is correct for a statistically significant portion of the subject (eg, a cohort in a cohort study). Whether a part is statistically significant is easily determined by those skilled in the art using a variety of well-known statistical evaluation tools such as confidence interval determination, p-value determination, Student's t-test, Mann-Whitney test, etc. Is possible. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001.

  In the context of the method of the present invention, the method shall diagnose whether the subject has early stage left ventricular hypertrophy (LVH). As used herein, the term “early-stage left ventricular hypertrophy” preferably relates to subtle structural changes in the myocardium that occur as it progresses from hypertension to heart failure. Therefore, it is possible to diagnose an intermediate phenotype as it progresses from hypertension to heart failure by performing the steps of the method described above. Thus, the term “early stage left ventricular hypertrophy” preferably includes a stage of LVH in which the left ventricular weight is slightly increased. Preferably, the left ventricular weight is slightly increased if the subject has LVMI as shown below.

Preferably, when the subject is a male, the term “early stage LVH” has a left ventricular weight index (and therefore the ratio of left ventricular weight to body surface, abbreviated LVMI) of 116 g / m ^{2 to} 149 g / range between m ^2, more preferably in the range between ^{116g / m 2 ~135g / m 2} , even more preferably in the range between 116g ^{/ m 2} ~130g ^/ m 2 and most preferably 116 g / ^{m 2,} comprising the stage of LVH ranges between ~125g / ^{m 2.}

Preferably, when the subject is female, the term “early stage LVH” has a left ventricular weight index (and thus the ratio of left ventricular weight to body surface, abbreviated LVMI) of 96 g / m ^{2 to} 125 g / range between m ^2, more preferably from ^96g / ^m 2 ^~120g / m ranges between ² or ^96g / m 2 ^~115g / range between ^{m 2,,} and most preferably ^96g / m 2 ^~108g / Includes the stage of LVH, which ranges between m ² .

Therefore, by performing the method of the present invention, it is possible to diagnose whether the subject to be tested has the left ventricular weight index as described above.
Preferably, the LVMI values provided herein are based on a method for assessing LVH as disclosed by Lang et al. (Recommendations for chamber quantification. Eur J Echocardiogr 2006; 7: 79-108).

In accordance with the above, the subject to be tested shall not suffer from a significant type of LVH. Preferably, if the subject is a male, the LVMI is lower than 149 g / m ² , lower than 135 g / m ² , lower than 130 g / m ² , or lower than 125 g / m ² (and therefore not higher). . Preferably, if the subject is female, the LVMI is lower than 125 g / m ² , lower than 120 g / m ² , lower than 115 g / m ² , or lower than 108 g / m ² (and therefore not higher). .

  The term “subject” in the context of the foregoing methods is an animal, preferably a mammal, and more preferably a human. In a preferred embodiment, the subject is male. In another preferred embodiment, the subject is female. Further, the subject can be older than 65 years.

  The subject preferably does not suffer from and / or does not show obvious symptoms of heart failure in the context of the aforementioned method of the invention (see description above). Thus, the subject shall not suffer from heart failure classified as stage C or D according to the ACC / AHA classification.

  As indicated above, subjects who do not exhibit obvious symptoms of heart failure can also suffer from stage A or B heart failure according to the ACC / AHA classification referred to above. In the context of the foregoing method of the invention, the subject thus preferably suffers from heart failure classified as stage A, or more preferably from stage B heart failure according to the ACC / AHA classification (J. Am. Coll. Cardiol. 2001; 38; 2101-2213, revised in 2005, J. Am.Coll.Cardiol.2005; 46; e1-e82).

  In another preferred embodiment of the foregoing method, the subject is healthy with respect to heart disease and disorders. A subject that is healthy with respect to heart disease and disorder is considered a subject not suffering from heart disease and disorder.

  In the context of the foregoing method, the subject shall have a retained left ventricular ejection fraction (LVEF). Thus, the subject should preferably have a left ventricular ejection fraction (LVEF) greater than 55%, more preferably greater than 60%, and most preferably greater than 65%. Thus, the subject preferably does not suffer from systolic heart failure or systolic dysfunction. The definition of the term “systolic heart failure” is provided above.

  The present invention is particularly suitable in subjects suffering from hypertension, which is a slight structural change in the myocardium that occurs as the combined determination of markers, endostatin and / or cardiac troponin progresses from hypertension to heart failure. This is to enable diagnosis. Thus, the subject preferably suffers from hypertension, especially arterial hypertension (see elsewhere herein for an explanation of this term).

  Further, it is envisaged that the subject does not suffer from ACS and / or coronary artery disease. In addition, the subject's renal function is not impaired (see above for definitions of these terms). Further, the subject shall not be pregnant. Again preferably the subject is not an athlete.

The terms “sample”, “determining”, “amount”, “compare” and “reference amount” are defined above. The definition also applies with respect to the method described above.
The definitions of the terms “cardiac troponin” and “BNP-type peptide” determined in the context of the method of the invention can also be found above. Preferred cardiac troponins are troponin T and troponin I. Preferred BNP-type peptides are NT-proBNP and BNP.

  The marker, endostatin, is well known in the art. Endostatin was originally isolated from murine hemangioendothelioma as a 20 kDa proteolytic fragment of type XVIII collagen (O'Reilly, MS et al., Cell 88 (1997) 277-285). Collagen represents a family of extracellular matrix proteins with a characteristic triple helix conformation that forms supramolecular aggregates and plays a major role in maintaining tissue structural integrity. Excess collagen deposition leads to fibrosis and destroys the normal function of surrounding tissues. Collagen XVIII is a member of the multiplex family of collagens with numerous interruptions in the central triple helix domain and a unique non-triple helix domain at the C-terminus, mainly in the basement membrane. The sequence of a short isoform of the human alpha 1 chain of collagen XVIII (SwissProt: P39060) is disclosed, for example, in WO2010 / 1224821, which is hereby incorporated by reference in its entirety.

  Endostatin is released from the alpha 1 chain of collagen XVIII by the action of various proteolytic enzymes (for details see Ortega, N. and Werb, Z., Journal of Cell Science 115 (2002) 4201-4214. See-the full disclosure of this paper is incorporated herein). Endostatin is suppressed herein by a collagen XVIII fragment spanning from amino acid 1337 to amino acid 1519 of collagen XVIII, as disclosed in WO2010 / 1224821. The C-terminal hinge region of the alpha chain of collagen XVIII contains several protease sensitive sites and many enzymes, including neutrophil elastase, cathepsin and matrix metalloproteinase, cleave the collagen chain at this region Is known to produce endostatin. These proteases do not exclusively release endostatin, but may also release other larger fragments containing endostatin sequences. As will be apparent to those skilled in the art, such larger fragments will also be measured by an immunoassay for endostatin.

  The term “reference amount” is defined elsewhere in this specification. As indicated above, the reference amount can also be a calculated reference amount. Preferably, the calculated reference amount should allow a distinction between subjects who are early stage LVH and subjects who are not early stage LVH.

  In principle, the following may apply as a diagnostic algorithm: Preferably, the test subject is (i) the amount of one of the determined markers (eg, endostatin or brain natriuretic peptide, or endostatin or cardiac troponin) Is higher than the reference amount, or (ii) if the amount of both markers is higher than the reference amount, then it is in the initial LVH. Preferably, the test subject is not in early stage LVH if the amount of both markers is lower than the reference amount.

  An appropriate reference amount that allows one to diagnose whether a subject is in early stage LVH can be determined by one skilled in the art. In the context of the foregoing method, a reference amount for a biomarker as referred to herein may be administered to a group of subjects in early stage left ventricular hypertrophy and / or a group of subjects not in early stage LVH. It is the amount that allows the body to be allocated. An appropriate threshold amount to separate the two groups is either in a subject or group of subjects known to be in early stage LVH, or in a subject or group of subjects known to be absent in early stage LVH Based on the amount of marker derived as referred to herein, it can easily be calculated by statistical tests referred to elsewhere in this specification.

  As indicated herein above, a reference amount of a biomarker can also be obtained from a subject or group of subjects known to be in early stage LVH. In this case, at least one amount of the determined marker that is essentially the same as or greater than the reference amount indicates that the subject is in an early stage LVH. Thus, if the amount of one marker is essentially the same as or greater than the reference amount, an early stage LVH can be diagnosed.

  Additionally or alternatively, the reference amount of the biomarker can be derived from a subject or group of subjects known not to be in early stage LVH. In this case, the amount of all markers that are essentially the same as the reference amount and / or lower than the reference amount (ie, the amount of both markers when determining two markers) It is not in LVH. Thus, if the amount of both markers is essentially the same as the reference amount and / or greater than the reference amount, the subject is not in early stage LVH.

Preferred reference amounts are in the range of 180-200 ng / ml for endostatin. A particularly preferred reference amount is 190 ng / ml.
A preferred reference amount of NT-proBNP is in the range of 180-250 pg / ml. A particularly preferred reference amount is about 200 pg / ml.

A preferred reference amount for troponin T is in the range of 5-10 pg / ml. Preferably, the reference amount is 8 pg / ml.
In an embodiment of the invention, the method further comprises recommending therapy if the subject is tested to be in early stage LVH. Preferred therapies are disclosed in the context of methods for diagnosing abnormal MFS.

  In a preferred embodiment of the present invention, the method comprises, in a sample from a subject, at least one additional marker selected from the group consisting of mimecan, growth differentiation factor 15 (GDF-15), FGF-23, and vitamin D. The method further includes determining an amount and comparing the amount (s) for the at least one additional marker thus determined to a reference amount (s) for the at least one additional marker. When determining the amount of FGF-23 and / or vitamin D, the ratio of FGF-23 to vitamin D (or vice versa) may be calculated and compared to a reference ratio. A preferred combination is cardiac troponin, endostatin and FGF-23: vitamin D.

  Mimecan is a small molecule proteoglycan containing leucine rich repeats and the precursor contains 298 amino acids. Other names for mimecan are OGN, osteoglycine, OG, OIF, SLRR3A.

  Mimecan is a member of the secreted small molecule leucine-rich proteoglycan (SLRP) family that contains structurally related core proteins. A common feature shared by all SLRPs is a tandem leucine rich repeat (LRP) unit in the C-terminal half of the core protein. However, in the N-terminal region, each class of SLRP has a unique domain containing a cysteine cluster with conserved spacing, termed the LRR N-domain. Class III SLRP contains six carboxyl LRRs and includes mimecan, epiphycan and opticin.

  Functional studies with mouse knockout of class I and II members such as decorin, biglycan, lumecan, and fibromodulin have shown a wide range of defects that can contribute to abnormal collagen fibril formation, and these SLRPs are Has been suggested to play an important role in establishing and maintaining (Ameye, L. and Young, MF, Glycobiology 12 (2002) 107R-116R). Class III mimecan failure also caused collagen fibrillary abnormalities (Tasheva, ES et al., Mol. Vis. 8 (2002) 407-415).

  Mimecan is a multifunctional component of the extracellular matrix. Mimecan is a variety of other proteins (IGF2, IKBKG, IFNB1, INSR, CHUK, IKBKB, NFKBIA, IL15, Cd3, retinoic acid, APP, TNF, lipopolysaccharide, c-abl oncogene 1, receptor tyrosine kinase, v- src sarcoma virus oncogene). These diverse binding activities can be a major cause of the ability of mimecan to perform diverse functions in many tissues.

  Mimecan can be found in the cornea, bone, skin, and additional tissues. Its expression pattern is altered in different pathological conditions. Despite the increasing amount of data on mimecan's biological role, its function remains unclear. Mimecan has been shown to be involved in controlling collagen fibril formation, an essential process in development, tissue repair, and metastasis (Tasheva et al., Mol. Vis. 8 (2002) 407-415). ). Mimecan, along with TGF-beta-1 or TGF-beta-2, plays a role in bone formation.

In an aspect of the present invention, a method for assessing whether a subject should be subjected to diagnostic assessment based on imaging or for establishing assistance to assess whether a subject suffers from abnormal MFS. There:
a) (i) contacting the sample with a detection agent that specifically binds to cardiac troponin for a time sufficient to allow formation of a complex of said detection agent and said cardiac troponin, and / or subjecting the sample to Contacting with a detection agent that specifically binds to FGF-23 for a time sufficient to allow formation of a complex of said detection agent and FGF-23, and (ii) the formed complex (single or The amount (s) of the complex (s) formed therein is determined by determining the amount (s) of cardiac troponin and / or FGF-23. And (iii) the amount (s) of the complex (s) formed reflects the amount (s) of cardiac troponin and / or FGF-23 present in the sample. , By converting the amount of a cardiac Troponin and / or FGF-23 (s), determining the amount of at least one marker cardiac troponin and / or FGF-23, it is selected from the group consisting of vitamin D;
b) comparing the amount (s) to the reference (s); and c) subjecting the subject to an imaging-based diagnostic evaluation based on the results of the comparison performed in step b) The method is intended to include assessing or establishing assistance to diagnose abnormal MFS.

In another aspect of the present invention, a system for assessing whether a subject should be subjected to an imaging-based diagnostic assessment, or for establishing assistance to diagnose abnormal MFS:
a) contacting the sample with a detection agent that specifically binds to cardiac troponin for a time sufficient to allow formation of a complex of said detection agent and cardiac troponin from the sample, and / or subjecting the sample to Contact with a detection agent that specifically binds to FGF-23 for a time sufficient to allow formation of a complex of said detection agent and FGF-23 from the sample (and optionally FGF-23 is determined) An analyzer unit configured to contact a detection agent that specifically binds to vitamin D and a sufficient time to allow formation of a complex of said detection agent and vitamin D from the sample. ,
b) an analyzer unit configured to measure the amount (s) of the complex (s) formed, said amount (s) of the complex (s) formed here Is proportional to the amount (s) of cardiac troponin and / or FGF-23 (and optionally vitamin D),
c) a computing device having a processor and in communication with said analyzer unit, and d) a non-transitory machine-readable medium comprising a plurality of instructions executable by the processor, wherein the instructions are executed When done, the amount (s) of cardiac troponin and / or FGF-23 (and optionally vitamin D) is determined by the amount of cardiac troponin and / or FGF-23 (and optionally vitamin D) in the sample (single or Convert the amount (s) of cardiac troponin and / or FGF-23 (and optionally vitamin D) to reflect the amount (s) and compare the amount (s) to the reference amount (s) And, based on the result of the comparison to the reference (s), subject is imaged The system is intended to include a medium for assessing whether it should be subjected to a diagnostic assessment or establishing assistance for diagnosing an abnormal MFS.

  A suitable detection agent, in one aspect, is an antibody that specifically binds to a marker, ie, an antibody that specifically binds to cardiac troponin or FGF-23, in a sample of a subject to be examined by the methods of the invention. It can also be an antibody that specifically binds. Another applicable detection agent can in one aspect be an aptamer that specifically binds to cardiac troponin or FGF-23. In yet another aspect, the sample is removed from the complex formed between the detection agents and before measuring at least the amount of complex formed. Thus, in one aspect, the detection agent may be immobilized on a solid support. In a further aspect, it is also possible to remove the sample from the complex formed on the solid support by applying a wash solution. The complex formed should be proportional to the amount of marker present in the sample. It will be appreciated that the specificity and / or sensitivity of the detection agent to be applied defines the degree of proportion of the marker contained in the specifically bindable sample. Further details of how the determination can be performed are also found elsewhere in this specification. The amount of complex formed shall be converted to the amount of at least one marker that reflects the amount actually present in the sample. Such an amount can, in one aspect, be essentially the amount present in the sample, or in another aspect, the amount of complex present and the amount present in the original sample. Depending on the relationship between them, it is also possible for the quantity to be that specific ratio.

  In a further aspect of the foregoing method, step a) may be performed by an analyzer unit, as defined in one aspect elsewhere in the specification.

  In the method aspect of the invention, the amount (s) determined in step a) are compared to a reference. In one aspect, the reference is a reference as defined elsewhere in this specification. In yet another aspect, the reference considers a proportional relationship between the amount of complex measured and the amount present in the original sample. Thus, the reference applied in the method aspect of the present invention is an artificial reference that has been adopted to reflect the limitations of the detection agent being used. In another aspect, the relationship may also include a normalization and / or correction calculation step when performing the comparison, eg, for the determined amount, before actually comparing the determined amount and the reference value, May be considered. Again, the normalization and / or correction calculation process for the determined amount employs a comparison process to properly reflect the limits of the detection agent that has been used. In one aspect, the comparison is performed automatically, for example with the aid of a computer system.

  In order to assist in evaluating whether the subject should be subjected to an imaging-based evaluation, in step b) the subject group to be subjected to the evaluation or to the subject group that should not be subjected to the evaluation Is established based on the comparison made. The assistance for diagnosing abnormal MFS is based on the comparison performed in step b) by assigning subjects to groups of subjects suffering from abnormal MFS or subjects not suffering from abnormal MFS. Established. As already discussed elsewhere in this specification, the subject assignments examined need not be correct in 100% of the examples examined. In addition, the subject group to which the examined subject is assigned is an artificial, established based on statistical considerations, i.e. a specific preselected degree of likelihood based on what the method of the invention will operate on. A group. In aspects of the invention, assistance is established automatically and is assisted, for example, by a computing device or the like as described and disclosed herein.

In a method aspect of the invention, the method further comprises recommending and / or managing the subject according to the results established in step c).
In the method aspect described above, steps b) and / or c) are performed by one or more analyzer units, as shown elsewhere herein.

  The invention also specifically relates to i) cardiac troponin and / or FGF-23, or ii) cardiac troponin in a sample of a subject for assessing whether the subject should be subjected to an imaging-based diagnostic assessment. It also relates to the use of a detection agent that binds and / or a detection agent that specifically detects FGF-23.

  When using FGF-23, the use may further include the use of vitamin D. Where the detection agent specifically binds to FGF-23, the use may further include the use of a detection agent that specifically binds vitamin D. In addition, the ratio between FGF-23 and vitamin D may be calculated.

  The present invention also provides i) cardiac troponin and / or FGF-23 (and for producing a pharmaceutical or diagnostic composition for assessing whether a subject should be subjected to imaging based diagnostic assessment. Use of vitamin D) or ii) a detection agent that specifically binds to cardiac troponin and / or a detection agent that specifically detects FGF-23 (and optionally a detection agent that specifically binds to vitamin D) Also related.

  The present invention also provides i) cardiac troponin and / or FGF-23, or ii) a detection agent and / or FGF-23 that specifically binds to cardiac troponin in a sample of a subject for diagnosing abnormal MFS. It also relates to the use of a detection agent that specifically detects.

  The present invention also provides i) cardiac troponin and / or FGF-23 (and optionally vitamin D) to produce a pharmaceutical or diagnostic composition for diagnosing whether a subject suffers from abnormal MFS. Or ii) the use of a detection agent that specifically binds to cardiac troponin and / or a detection agent that specifically detects FGF-23 (and optionally a detection agent that specifically binds vitamin D). A preferred detection agent is an antibody that specifically binds to the marker to be measured.

  When using FGF-23, the use may further include the use of vitamin D. When using a detection agent that specifically binds to FGF-23, the use may further include the use of a detection agent that specifically binds to vitamin D. In addition, the ratio between FGF-23 and vitamin D may be calculated.

  The present invention also provides i) FGF-23 and / or IGFBP7 (and optionally cardiac troponin and / or brain natriuretic peptide) in a sample of a subject for predicting the risk of death and / or cardiovascular events, or ii) a detection agent that specifically binds to FGF-23 and / or a detection agent that specifically detects IGFBP7 (and optionally a detection agent that specifically binds to cardiac troponin and specifically to brain natriuretic peptide) It also relates to the use of detection agents to be detected.

  The present invention also provides i) FGF-23 and / or IGFBP7 (and optionally cardiac troponin and / or for the manufacture of a pharmaceutical or diagnostic composition for predicting the risk of death and / or cardiovascular events. Brain natriuretic peptide), or ii) a detection agent that specifically binds to FGF-23 and / or a detection agent that specifically detects IGFBP7 (and optionally a detection agent and brain that specifically binds to cardiac troponin) It also relates to the use of a detection agent that specifically detects natriuretic peptides.

  When using FGF-23, the use may further include the use of vitamin D. When using a detection agent that specifically binds to FGF-23, the use may further include the use of a detection agent that specifically binds to vitamin D. In addition, the ratio between FGF-23 and vitamin D may be calculated.

  The present invention also specifically binds i) endostatin and BNP-type peptides, or ii) endostatin in samples of subjects with retained left ventricular ejection fraction for diagnosing early stage LVH It also relates to the use of detection agents and / or detection agents that specifically bind to BNP-type peptides.

  The present invention also provides i) endostatin for the manufacture of a pharmaceutical or diagnostic composition for diagnosing whether a subject has retained left ventricular ejection fraction to diagnose early stage LVH It also relates to the use of a BNP-type peptide, ii) a detection agent that specifically binds to endostatin and / or a detection agent that specifically binds to a BNP-type peptide. A preferred detection agent is an antibody that specifically binds to the marker to be measured.

  The present invention also provides detection that specifically binds i) endostatin and cardiac troponin, or ii) endostatin in a sample of a subject with retained left ventricular ejection fraction to diagnose early stage LVH. It also relates to the use of agents and detection agents that specifically bind to cardiac troponin.

  The invention also provides i) endostatin and cardiac troponin for the manufacture of a pharmaceutical or diagnostic composition for diagnosing early stage LVH in a subject with retained left ventricular ejection fraction, or ii) It also relates to the use of a detection agent that specifically binds to endostatin and a detection agent that specifically binds to cardiac troponin. A preferred detection agent is an antibody that specifically binds to the marker to be measured.

  The present invention is also specific to i) endostatin, brain natriuretic peptide and cardiac troponin, or ii) endostatin in samples of subjects with retained left ventricular ejection fraction for diagnosing early stage LVH It also relates to the use of detection agents that specifically bind, detection agents that specifically bind to BNP-type peptides, and detection agents that specifically bind to cardiac troponin.

  The term “detection agent” as used herein refers to an agent capable of specifically recognizing and binding to biomarker polypeptide (s) present in a sample. The term is used herein interchangeably with the term “ligand”. Furthermore, the agent shall allow direct or indirect detection of the complex formed by the agent and the biomarker. Direct detection can be achieved by including a detectable label in the agent. Indirect labeling can be achieved by an additional agent that specifically binds to the complex comprising the biomarker and the detection agent, which can then produce a detectable signal. Suitable compounds that can be used as detection agents are well known in the art. Preferably, the detection agent is an antibody, in particular a monoclonal antibody, or an aptamer that specifically binds to a biomarker as referred to herein. The term “antibody” is defined elsewhere in this specification.

According to a preferred embodiment of the present invention, a device adapted for carrying out the method of the present invention, comprising: a) a detection agent that specifically binds to cardiac troponin and / or a detection agent that specifically binds to FGF-23 (As well as, optionally, when determining FGF-23, an analyzer unit comprising a detection agent that specifically binds vitamin D), wherein the amount (s) of marker (s) in a subject sample is determined Said unit adapted to determine; and b) compare the determined amount (s) with the reference amount (s), thereby assessing whether the subject should be subjected to imaging based assessment Analyzer unit for a database containing reference quantity (s) and a computer for performing a comparison Including instrumentation algorithm, including the unit, to provide the device.

Preferred reference quantities and algorithms are disclosed elsewhere herein.
According to a preferred embodiment of the present invention, a device adapted for carrying out the method of the present invention, comprising: a) a detection agent that specifically binds to cardiac troponin and / or a detection agent that specifically binds to FGF-23 (As well as, optionally, when determining FGF-23, an analyzer unit comprising a detection agent that specifically binds vitamin D), wherein the amount (s) of marker (s) in a subject sample is determined Said unit adapted to determine; and b) to compare the determined amount (s) with the reference amount (s), thereby assessing whether the subject suffers from abnormal MFS An analyzer unit, a database containing reference quantity (s) and a computer-implemented algorithm for performing comparisons Including prism, including the unit, to provide the device.

According to another preferred embodiment of the present invention, a device adapted for carrying out the method of the present invention, comprising a) a detection agent that specifically binds to IGFBP7, and / or a detection that specifically binds to FGF-23. An analyzer unit comprising an agent (and optionally a detection agent that specifically binds to cardiac troponin and / or a detection agent that specifically binds to brain natriuretic peptide), wherein the marker (s) in the subject sample is ) Said unit adapted to determine the amount (s); and b) comparing the determined amount (s) with the reference amount (s), thereby causing death and / or heart An analyzer unit for predicting the risk of vascular events, including a reference quantity (s) , And a computer-implemented algorithm for performing the comparison, including the unit, to provide the device.

In accordance with another preferred embodiment of the present invention, a device adapted to carry out the method of the present invention, comprising a) i) a detection agent that specifically binds to endostatin, and ii) specifically to a BNP-type peptide. An analyzer unit comprising a detection agent that binds and / or a detection agent that specifically binds to cardiac troponin, i) endostatin in a sample from a subject having retained LVEF, and ii) a BNP-type peptide and Said unit adapted to determine the amount of cardiac troponin; and b) an analyzer unit for diagnosing early left ventricular hypertrophy by comparing the determined amount (s) with a reference amount A database containing a reference quantity for the marker and a computer implementation for performing the comparison Including algorithms, including the unit, to provide the device.

Preferred reference quantities and algorithms are disclosed elsewhere herein.
When determining FGF-23 and vitamin D, the ratio shall be calculated as indicated elsewhere in this specification and compared to a reference ratio. In this case, an analyzer unit as shown in the above device context shall (again) make it possible to calculate the ratio between FGF-23 and vitamin D. Further, the database shall include reference ratio values.

Preferred embodiments of the present disclosure include a system for assessing whether a subject should be subjected to imaging-based assessment, or for diagnosing abnormal MFS, as shown elsewhere herein. . Examples of systems include clinical chemistry analyzers, coagulation chemistry analyzers, immunochemical analyzers that are used to detect the results of chemical or biological reactions or to monitor the progress of chemical or biological reactions. Urine analyzer, nucleic acid analyzer. More specifically, exemplary systems of the present disclosure include the Roche Elecsys ^™ system and Cobas® e immunoassay analyzer, Abbott Architect ^™ and Axym ^™ analyzers, Siemens Centaur ^™ and Immulite ^™ analyzers, and Beckman Coulter Celter. ^TM and Access ^TM analyzers can also be included.

  System aspects may include one or more analyzers utilized to implement the present disclosure. The analyzer unit of the system disclosed herein is in communication with any computing device disclosed herein via any wired communication, Bluetooth, LANS, or wireless signal, as is known. Furthermore, in accordance with the present disclosure, the analyzer unit may comprise a stand-alone device or module in a larger instrument that performs one or both detections, eg, qualitative and / or quantitative evaluation of a sample for diagnostic purposes. It can also be included. For example, the analyzer unit may perform or assist in pipetting, dispensing, mixing of samples and / or reagents. The analyzer unit can also include a reagent holding device for holding the reagent and performing the assay. Reagents may be placed in the form of containers or cassettes containing individual reagents or reagent groups, for example, placed in appropriate storage locations or locations within a storage compartment or conveyor. The detection reagent may also be of the type immobilized on a solid support, where the sample is contacted. In addition, the analyzer unit may include processes and / or detection components that can be optimized for a particular analysis.

  According to some embodiments, the analyzer unit can also be configured for optical detection of an analyte, such as a marker, in a sample. An exemplary analyzer unit configured for optical detection includes a device configured to convert electromagnetic energy into an electrical signal, the device including both single and multi-element or array optical detectors . In accordance with the present disclosure, the optical detection device monitors the optical electromagnetic signal and is an electrical exit signal compared to a baseline signal that is indicative of the presence and / or concentration of the analyte in a sample located in the optical path. Or it is possible to provide a reaction signal. Such devices also include, for example, photodiodes, including avalanche photodiodes, phototransistors, photoconductive detectors, linear sensor arrays, CCD detectors, CMOS detectors including CMOS array detectors, photomultiplier tubes, and A photomultiplier tube array may also be included. According to certain embodiments, the light detection device, such as a photodiode or photomultiplier, may contain additional signal conditioning or processing electronics. For example, the optical detection device may include at least one preamplifier, an electronic filter, or an integrated circuit. Suitable preamplifiers include, for example, integrated, transimpedance, and current amplification (current mirror) preamplifiers.

  Further, one or more analyzer units according to the present disclosure may include a light source for light emission. For example, the light source of the analyzer unit is at least one light emission for measuring the analyte concentration in the sample to be tested or for allowing energy transfer (eg through fluorescence resonance energy transfer or enzyme catalysis). It can also consist of elements (eg light emitting diodes, power radiation sources such as incandescent lamps, electroluminescent lamps, gas discharge lamps, high intensity discharge lamps, lasers).

  Further, the analyzer unit of the system may include one or more incubation devices (eg, to maintain a sample or reagent at a specific temperature or temperature range). In some embodiments, the analyzer unit may include a thermocycler, including a real-time thermocycler, to subject the sample to repeated temperature cycles and monitor changes in the amount of amplification product in the sample. May be.

  Further, the analyzer unit of the system disclosed herein may include or be operably coupled to a reaction vessel or cuvette supply unit. Exemplary supply units include liquid processing units, such as pipetting units, that deliver samples and / or reagents to reaction vessels. The pipetting unit may include a reusable washable needle, such as a steel needle, or a disposable pipette tip. The analyzer unit may further comprise one or more mixing units, for example a shaking unit for shaking the cuvette containing the liquid, or a mixing paddle for mixing the liquid in the cuvette, or a reagent container.

  From the above, according to some aspects of the present disclosure, some of the steps of the methods disclosed and described herein may be performed by a computing device. The computing device can be, for example, a general purpose computer or a portable computing device. It should also be understood that multiple computing devices may be used together to perform one or more steps of the methods disclosed herein, either through a network or through other methods of transferring data. . Exemplary computing devices include desktop computers, laptop computers, personal data assistants (“PDAs”), such as BLACKBERRY brand devices, cellular devices, tablet computers, servers, and the like. Generally, a computing device includes a processor capable of executing a plurality of instructions (for example, a software program).

  The computing device has access to the memory. The memory is a computer-readable medium and may include a single storage device or multiple storage devices, eg, located locally with the computing device or accessible to the computing device over a network. Computer readable media can be any available media that can be accessed by a computing device and includes both removable and non-removable media. In addition, computer readable media can be one or both of removable media and non-removable media. For example, and without limitation, computer-readable media can also include computer storage media. Exemplary computer storage media include, but are not limited to, RAM, ROM, EEPROM that can be used to store a plurality of instructions that are accessible by a computing device and executable by a processor of the computing device. Flash memory or any other memory technology, CD-ROM, digital versatile disk (DVD) or other optical disk storage, magnetic cassette, magnetic tape, magnetic disk storage or other magnetic storage device, or any other Media included.

  In accordance with aspects of the present disclosure, the software may include instructions that, when executed by the processor of the computing device, can perform one or more steps of the methods disclosed herein. Some of the instructions may be adapted to produce signals that control the operation of other machines, and therefore can operate through these control signals to convert material far away from the computer itself. Is possible. These descriptions and representations are the means used by those skilled in the data processing arts and, for example, most effectively convey the contents of their work to others skilled in the art.

  The plurality of instructions may also include an algorithm that is generally assumed to be a consistent sequence of steps leading to a desired result. These steps are those requiring physical treatment of physical quantities. Usually, though not necessarily, these quantities take the form of electrical or magnetic pulses or signals capable of being stored, transferred, converted, combined, compared, and otherwise processed. . Sometimes, in principle, for general use, these signals are referred to by values, letters, display data, numbers, etc. in relation to the physical item or manifestation in which such signals are embodied or expressed. It turns out that it is suitable. However, it should be borne in mind that all of these and similar terms are to be associated with the appropriate physical quantities and are merely used herein as suitable labels applied to these quantities. In accordance with some aspects of the present disclosure, an algorithm for performing a comparison between the determined amount of one or more markers disclosed herein and an appropriate reference is implemented by performing the instructions. And executed. The results may be provided as output of parametric diagnostic raw data, or as absolute or relative quantities. In accordance with various aspects of the system disclosed herein, “diagnosis” can be provided by the computing device of the system disclosed herein based on the comparison of the calculated “amount” to a reference or threshold. It is. For example, the computing device of the system may provide an indicator in the form of words, symbols, or numbers that are indicators of a particular diagnosis.

  The computing device can also have access to the output device. Exemplary output devices include, for example, fax machines, displays, printers, and files. In accordance with some aspects of the present disclosure, the computing device may also perform one or more steps of the methods disclosed herein, and then through the output device, the result of the method, the indication, It is also possible to provide an output related to the ratio or other factors.

  Finally, the present invention includes a detection agent that specifically binds to cardiac troponin and / or a detection agent that specifically binds to FGF-23, a reference standard, and instructions for performing the method, It relates to a kit adapted to carry out the method of the invention.

In a preferred embodiment, the kit may further comprise a detection agent that specifically binds vitamin D.
The term “kit” as used herein is a collection of the aforementioned components, preferably provided separately or in a single container. The container also includes instructions for performing the method of the present invention. These instructions can be in the form of a manual or can be compared as mentioned in the method of the invention and, when implemented on a computer or data processing device, as appropriate. It may be provided by computer program code capable of establishing a diagnosis. The computer program code may be provided on a data storage medium or device, such as an optical storage medium (eg, a compact disc), or directly on a computer or data processing device. In addition, the kit shall include at least one standard for reference as defined above.

  In some embodiments, the kits disclosed herein include at least one component or packaged combination of components for performing the disclosed methods. By “packaged combination”, a kit can include one or more components, eg, probes (eg, antibodies), controls, buffers, reagents (eg, conjugates and / or substrates) as disclosed herein. ), Which means providing a single package containing instructions for use. Kits containing a single container are also included within the definition of “packaged combination”. In some embodiments, the kit includes at least one probe, such as an antibody (having specific affinity for an epitope of a biomarker as disclosed herein). For example, the kit may include an antibody labeled with a fluorophore, or an antibody that is a member of a fusion protein. In the kit, the probe may be immobilized and may be immobilized in a specific conformation. For example, an immobilized probe can be provided in the kit to specifically bind to the target protein, detect the target protein in the sample, and / or remove the target protein from the sample.

  According to some embodiments, the kit includes at least one probe that may be immobilized in at least one container. The kit may also include a number of optionally immobilized probes in one or more containers. For example, multiple probes may be present in a single container or in separate containers, for example, each container may contain a single probe.

  In some embodiments, the kit may include one or more non-immobilized probes and one or more solid supports with or without immobilized probes. Some such embodiments may be used to bind the immobilized probe to some or all of the reagents and supplies necessary to immobilize one or more probes to a solid support, or to a specific protein in a sample. May include some or all of the reagents and supplies required for

  In certain embodiments, a single probe (including multiple copies of the same probe) may be immobilized on a single solid support and provided in a single container. In other embodiments, two or more probes, each specific for a different target protein or a different type (eg, a particular epitope) of a single target protein, may be provided in a single container. In some such embodiments, immobilized probes may be provided in a number of different containers (eg, in a single use form), or a number of immobilized probes are provided in a number of different containers. May be. In further embodiments, the probe may be immobilized on a number of different types of solid supports. Any combination of immobilized probe (s) and container (s) is contemplated in the kits disclosed herein, and any combination can be selected to produce a kit suitable for the desired use. It can also be achieved.

  The container of the kit may package and / or package one or more components disclosed herein, including, for example, probes (eg, antibodies), controls, buffers, and reagents (eg, conjugates and / or substrates). It may be any container suitable for containing. Suitable materials include, but are not limited to, glass, plastic, cardboard or other paper products, wood, metal, and any alloys thereof. In some embodiments, the container may completely enclose the fixed probe (s) or simply cover the probe to minimize contamination by dust, oil, etc., and exposure to light. May be. In some further embodiments, the kit may include a single container or multiple containers, and if multiple containers are present, each container may be the same as all other containers, It may be different, or different from some but not different from all other containers.

The preferred embodiments of the present invention are listed below. The definitions and descriptions provided above in the specification and in the claims apply mutatis mutandis.
1. A method for assessing whether a subject should be subjected to diagnostic assessment based on imaging, comprising: a) in a sample from a subject, of cardiac troponin and / or fibroblast growth factor 23 (FGF-23) Should the amount (s) be determined, and b) whether the thus determined amount (a) is compared to the reference amount (s), thereby subjecting the subject to imaging-based diagnostic evaluation Evaluating the method.

2. The method of embodiment 1, wherein the subject suffers from hypertension.
3. The method of aspects 1 and 2, wherein the subject does not suffer from left ventricular hypertrophy, in particular the subject has a normal left ventricular weight.

4). The method according to any one of aspects 1-3, wherein the diagnostic assessment based on imaging is echocardiography or magnetic resonance imaging.
5). The method according to any one of aspects 1 and 4, wherein the diagnostic assessment based on imaging is for diagnosing diastolic dysfunction and / or systolic dysfunction, in particular for diagnosing an abnormal medial wall shortening rate. .

6). The method of any one of aspects 1-5, wherein an increase in the amount in the sample from the subject when compared to the reference amount indicates that the subject should be subjected to diagnostic assessment based on imaging.
7). The method of any one of aspects 1-6, wherein a decrease in the amount in the sample from the subject when compared to the reference amount indicates that the subject should not be subjected to diagnostic assessment based on imaging.

8). The method according to any one of aspects 1 to 7, wherein the reference amount is a calculated reference amount.
9. The reference amount is the amount of cardiac troponin and / or FGF-23 in the first sample obtained from the subject before the sample (“second sample”) as shown in step a) of embodiment 1. A method according to any one of embodiments 1-7.

10. The method of embodiment 9, wherein the first sample is obtained 6-18 months prior to the second sample.
11. At least 10%, or in particular at least 25% more than the amount in the first sample, the amount of cardiac troponin, in particular troponin T, in the second sample, and / or at least 5%, or in particular at least at least the amount in the first sample. The method of embodiment 9 or 10, wherein the amount of FGF-23 in the second sample, which is 10% greater, is an indicator of the subject to be subjected to diagnostic assessment based on imaging.

12 The method according to any one of aspects 9 to 11, wherein the subject did not suffer from abnormal MFS when the first sample was obtained.
13. The method according to any one of aspects 9 to 12, wherein the subject suffered from hypertension when the first sample was obtained.

14 A method for diagnosing abnormal medial wall shortening rate (MFS) in a subject comprising: a) determining the amount of cardiac troponin and / or fibroblast growth factor 23 (FGF-23) in a sample from the subject And b) comparing the amount thus determined to a reference amount, thereby diagnosing an abnormal medial wall shortening rate.

15. The method of embodiment 14, wherein the subject does not suffer from left ventricular hypertrophy, in particular the subject has a normal left ventricular weight.
16. An increase in the amount in the sample from the subject compared to the reference amount indicates that the subject suffers from abnormal MFS, and a decrease in the amount in the sample from the subject compared to the reference amount is The method of aspects 14 and 15, wherein the subject indicates that it does not suffer from abnormal MFS.

17. The method according to any one of aspects 1 to 16, wherein the sample is a blood, serum or plasma sample.
18. The method of any of embodiments 1-17, wherein the amounts of both cardiac troponin and FGF-23 are determined.

  19. In determining the amount of FGF-23, the method determines in step a) in addition to the determination of FGF-23, the amount of vitamin D in a further step a1), the amount of FGF-23 relative to the amount of vitamin D. A method according to any one of embodiments 1-18, comprising calculating a ratio of the ratio and comparing the ratio thus calculated to a reference ratio.

20. The method of any one of aspects 1-19, wherein the subject does not suffer from renal dysfunction.
21. 21. The method of any one of aspects 1-20, wherein the subject does not have increased NT-proBNP levels, particularly in blood, serum or plasma samples.

  22. A detection agent that specifically binds to i) cardiac troponin and / or FGF-23 or ii) cardiac troponin in a subject sample for assessing whether the subject should be subjected to imaging based diagnostic assessment And / or use of a detection agent that specifically binds to FGF-23.

23. A device adapted to perform the method of aspects 1-13, comprising: a) a detection agent that specifically binds to cardiac troponin, and / or a detection agent that specifically binds to FGF-23 (and optionally FGF To determine -23, an analyzer unit comprising a detection agent that specifically binds vitamin D) to determine the amount (s) of the marker (s) in the subject sample Said adapted unit; and b) an analyzer unit for comparing the determined quantity (s) with the reference quantity (s) and thereby assessing whether the subject is to be subjected to an imaging-based assessment Including a database containing the reference quantity (s) and a computer-implemented algorithm for performing the comparison , Including the unit, the device.

  All references cited in this specification are hereby incorporated by reference in their entirety, as well as the disclosure content specifically mentioned in this specification.

The following examples merely illustrate the invention. These should not be considered as limiting the scope of the invention in any way.
Example 1: Patient cohort FGF-23 (ELISA, Immupics), total 25-hydroxyvitamin D (ECLIA, Roche Diagnostics), IFGBP7, endostatin, mimecan and two heart markers (hs-cTnT and NT-proBNP, Roche Elecsys assay) plasma levels and between elevated LV weight / BSA (> 95 g / m ^{2 for} women,> 115 for men), subnormal midwall shortening (MFS <15%) and death Was investigated in 2001 elderly people (average age 73 ± 5 years, 48% women) living in Central Italy (PREDICTOR study). Subjects were divided into four categories according to LV weight index and normal values of MF. Markers in plasma samples were determined. Data on deaths from all causes of death were available for a subgroup of 1200 subjects after a median follow-up of 47 months (86 deaths).

  Three markers (endostatin, IGFBP7 mimecan / osteoglycine) were examined in 550 elderly subjects, selected from 2001 subjects aged 65-84 years of the epidemiological study PREDICTOR. This sub-cohort includes 50 normal subjects, 150 AHA / ACC stage A subjects, 300 stage B subjects. The median follow-up was 47 months (21 deaths).

Subjects were divided into four categories according to LV weight index and normal values of MFS.
a) Subjects with normal LV weight index (LV weight / BSA ≦ 95 g / m ^{2 for} women and ≦ 115 g / m ^{2 for} men) and normal MFS (≧ 15%) b) Normal LV weight index ( Subjects with LV weight / BSA ≦ 95 g / m ^{2 for} women and ≦ 115 g / m ^{2 for} men) and abnormal MFS (<15%) c) elevated LV weight index (LV weight for women / BSA> 95 g / m ² , and> 115 g / m ^{2 for} men) and normal MFS (≧ 15%) d) Elevated LV weight index (LV weight / BSA> 95 g / m for women) ^2, and for men> 115 g / ^{m 2)} and abnormal MFS (<subjects example 2 with 15%): assay Roche electrochemiluminescence E ISA sandwich test Elecsys Troponin T hs (high sensitivity) STAT (short turnaround time) assay was used to determine troponin T. The test uses two monoclonal antibodies specifically directed against human cardiac troponin T. The antibody recognizes two epitopes (amino acids 125-131 and 136-147) located in the central part of the 288 amino acid cardiac troponin T protein. The hs-TnT assay allows measurement of troponin T levels in the range of 3 to 10000 pg / mL.

  NT-proBNP was determined using the Roche electrochemiluminescence ELISA sandwich test Elecsys pro BNP II STAT (short turnaround time) assay. The test uses two monoclonal antibodies that recognize an epitope located in the N-terminal part (1-76) of proBNP (1-108).

  Immunotopics Inc. , Catalog number 60-6100, by using the (C-terminal) ELISA kit of the second generation enzyme-linked immunosorbent assay (ELISA) to determine human fibroblast growth factor 23 levels in plasma or cell culture medium. FGF-23 was determined. This second generation human FGF-23 (C-terminal) ELISA kit is a two-site enzyme linked immunosorbent assay (ELISA) for the measurement of FGF-23 in plasma or cell culture media. Two affinity-purified goat polyclonal antibodies have been selected to detect epitopes within the carboxyl-terminal (C-terminal) portion of FGF-23. One antibody is biotinylated for capture and the other antibody is conjugated with an enzyme, horseradish peroxidase (HRP) for detection. These antibodies bind to both an intact molecule and a large carboxyl terminal fragment of human FGF-23. Samples containing human FGF-23 are incubated simultaneously in biotinylated capture antibody and HRP-conjugated antibody in microtiter wells coated with streptavidin. FGF-23 contained in the sample is immunologically bound by a capture antibody and a detection antibody to form a “sandwich” complex. At the end of the incubation period, the wells are washed to remove any unbound antibody and other components. The enzyme bound to the wells is incubated with the substrate solution in a timed reaction and then measured in a spectrophotometric microtiter plate reader. The enzyme activity of the antibody complex bound to the well is directly proportional to the amount of FGF-23 in the sample. A standard curve is generated by plotting the absorbance against the respective FGF-23 concentration for each standard on a linear or log scale. The concentration of human FGF-23 in the sample is determined directly from this curve.

  A sandwich ELISA was used for detection of mimecan in human serum or plasma. An aliquot of anti-mimecan polyclonal antibody from R & D Systems (catalog number: AF2660) is conjugated with biotin and digoxigenin, respectively, for antigen capture and detection. Streptavidin-coated 96-well microtiter plates are incubated with 100 μl biotinylated anti-mimecan polyclonal antibody for 60 minutes at 0.2 μg / ml in 1 × PBS solution. After incubation, the plates were washed 3 times with 1 × PBS + 0.02% Tween-20, blocked with PBS + 2% BSA (bovine serum albumin) for 45 minutes and then again washed 3 times with 1 × PBS + 0.02% Tween-20. The wells are then incubated with either 100 μl of serial dilutions of recombinant mimecan as standard antigen, or diluted serum or plasma samples (1: 5 in 1 × PBS + 1% BSA) from patients or control individuals, respectively. After binding of mimecan, the plate is washed 3 times with 1 × PBS + 0.02% Tween-20. For specific detection of bound mimecan, wells are incubated with 100 μl of digoxigenated anti-mimecan polyclonal antibody for 45 minutes at 0.2 μg / ml in 1 × PBS + 1% BSA. The plate is then washed 3 times to remove unbound antibody. In the next step, the wells are incubated with 100 μl of 75 mU / ml anti-digoxigenin-POD conjugate (Roche Diagnostics GmbH, Mannheim, Germany, catalog number 1633716) for 30 minutes in 1 × PBS + 1% BSA. The plate is subsequently washed 6 times with the same wash buffer as above. For detection of antigen-antibody complexes, the wells were incubated with 100 μl of ABTS solution (Roche Diagnostics GmbH, Mannheim, Germany, catalog number 11685767) and the optical density (OD) at 405 and 492 nm after 15 min. Measure using an ELISA reader.

  A commercially available sandwich ELISA (Quantikine human endostatin immunoassay, catalog number DNSTO, R & D Systems) was used for the determination of endostatin in human serum or plasma. Perform the measurement according to the instructions provided by the manufacturer.

  A sandwich ELISA was used for detection of IGFBP7 in human serum or plasma. An aliquot of R & D Systems' anti-IGFBP7 polyclonal antibody (Cat. No. AF1334) was conjugated with biotin and digoxigenin, respectively, for antigen capture and detection.

Example 3: Results
Table 1 shows the results of slight modification of the LV shape-shortening of the inner wall . Median concentrations of hs-cTnT, NT-proBNP, FGF-23 and vitamin D were low on average. Median concentrations of hs-cTnT, Nt-proBNP, and FGF-23 / vitamin D are higher in subjects with elevated LV weight and / or subnormal MFS (both adjusted for gender and age) it was high. FGF-23 and vitamin D were significantly related to LV weight, independent of hs-cTnT.

  Table 1: LV weight index / MFS category

^* P Adjusted for gender and age. ^# Ratio between circulating levels of FGF-23 and vitamin D (calculated individually for each subject)
Median NT-proBNP concentrations did not increase in older subjects with normal LV weight index and subnormal MFS versus older subjects with normal LV weight and normal MFS.

  In contrast, the median concentration of hs-cTnT, and the median concentration ratio of FGF-23 / vitamin D, were compared to normal LV weight index and subnormal MFS for elderly subjects with normal LV weight and normal MFS. In elderly subjects with a significant increase, 37% and 11%.

  Cardiac troponin and FGF-23 levels are elevated in elderly subjects, with slight alterations in LV shape and function (even before detectable alterations in LV shape). Thus, the determination of these markers will help identify intermediate phenotypes in the progression from hypertension to systolic or diastolic dysfunction (by echo, MRI, ECG) before LVH is revealed. . The ratio of FGF-23 / vitamin D may be further determined. An increase in the level of a marker selected from cardiac troponin, FGF-23 (preferably FGF-23 / vitamin D ratio) compared to the respective reference level indicates that the patient suffers from MFS.

Death / cardiovascular events NT-proBNP, hs-cTnT, vitamin D and FGF-23 were determined in 1202 subjects and the data included all causes of death. The median follow-up was 47 months [Q1-Q3 39-55]. The results are shown in the table below. Continuous data are shown as mean ± SD excluding circulating biomarkers (median [Q1-Q3]). Let category data be n (%).

  Marker according to survival status (death vs. survival)

  The following data provides a more comprehensive overview (and includes, for example, clinical characteristics).

  As can be seen from the table, 86 subjects (7.1%) died during approximately 4 years of follow-up. Plasma concentrations of hs-cTnT, NT-proBNP and FGF-23 were higher than the dead, while vitamin D was lower.

  The table below provides a univariate Cox analysis (HR: hazard ratio) for the association between biomarker concentrations and all causes of death.

As can be seen from the table, NT-proBNP, hs-cTnT, vitamin D and FGF-23 are significantly associated with all causes of death in a univariate analysis.
The table below provides a multivariate Cox analysis for the adjusted association between biomarker concentrations and all causes of death.

  Thus, higher circulating levels of FGF-23 (HR 1.88 [1.40-2.54], p <0.0001) and lower concentrations of vitamin D (HR 0.68 [0.50-0. 93], p = 0.02) is associated with all causes of death after adjusting for demographic and clinical variables and for reference cardiac biomarkers (NT-proBNP or hs-cTnT) (86 Death, 7.2%).

  In addition, the markers IGFPB7, mimecan and endostatin were assayed in samples from 550 elderly subjects selected from the Predictor study. The results are shown in the table below. Excluding circulating biomarkers, continuous data are shown as mean ± SD (median [Q1-Q3]). Let category data be n (%).

  Marker according to survival status (dead vs survivor)

  The following table provides a more comprehensive overview (and includes, for example, clinical characteristics):

  Comment: As can be seen from the table, the median LVEF was greater than 65%. The tested subjects were therefore not suffering from systolic heart failure. Twenty-one subjects (4.1%) died during approximately 4 years of follow-up. The median LVEF for the dead was> 60%. Plasma concentrations of all biomarkers IGFBP7 were significantly higher in the dead, but mimecan and endostatin were not.

  The table below provides a univariate Cox analysis for the association between biomarker concentrations and all causes of death (HR: hazard ratio).

  NT-proBNP, hs-cTnT and IGFBP7 are significantly associated with all causes of death in a univariate analysis, but mimecan and endostatin are not. Interestingly, the maximum hazard ratio is observed with IGFBP7. Thus, IGFBP7 is a better predictor of mortality risk than troponin T or NT-proBNP.

  The following table provides a multivariable Cox analysis for the association between adjusted biomarker concentrations and all causes of death.

After adjustment, NT-proBNP, hs-cTnT and IGFBP7 are significantly associated with all causes of death. A maximum hazard ratio is observed for IGFBP7.
Detection of slight cardiac changes Seven markers (NT-proBNP, hsTnT, endostatin, IGFBP7 mimecan / osteoglycine, FGF-23, vitamin D) were examined in 550 elderly subjects selected from the PREDICTOR study. The clinical characteristics of this subcohort are described above. In subjects with slight myocardial changes, LVH and / or decreased MFS and retained ejection fraction (LVEF> 55%), cardiac troponin, FGF-23, IGFBP7, endostatin and mimecan levels are elevated On the other hand, vitamin D levels decrease. Thus, the determination of these markers will help identify the median phenotype as it progresses from hypertension to heart failure. It is also possible to further determine the ratio of FGF-23 / vitamin D. Increased levels of markers selected from NT-proBNP, endostatin, cardiac troponin or from FGF-23 (preferably FGF-23 / vitamin D ratio) and mimecan compared to their respective reference levels Shows suffering slight heart changes, MFS and / or LVH.

The table below shows the sensitivity of 80% specificity of NT-proBNP or cardiac troponin T and endostatin combination in detecting samples of early HF stage (n = 352) in the 550 patient sub-cohort of the Predictor study. provide. Samples from borderline LVH or mildly elevated LV weight (96-121 g / m ² LV weight female; 116-148 g / m ² LV weight male) or abnormal MFS or stage C patients. The control group consisted of HF stage 0 or A patient samples (n = 211).

As can be seen from the table, for both markers alone, the combination of NT-proBNP or cTnThs and endostatin caused slight cardiac changes, LVH, abnormal MFS, early heart failure stage B and stage C. Detect a higher rate of patients with it. As can be obtained from the table, (in women, 95~132g / ^{m 2,} and in the case of men, 116~148g / ^{m 2)} moderate abnormal LV weight from mild borderline several patients of , NT-proBNP (239/352) or cTnT (211/352) or endostatin (211/352) alone in the control group (stage 0 or A 211 with no slight myocardial changes) Not detected with 80% specificity in the sample).

  The combination of the two markers was 80% specific, with only 43% (endostatin and NT-proBNP) of 32% (NT-proBNP) and 40% (endostatin) of both markers alone. Resulted in improved sensitivity. The combination of the two markers is 80% specific and an even more improved sensitivity of 47% (endostatin and cTnThs) versus 40% (cTnThs) and 40% (endostatin) of both markers alone Produced. The combination of cTnT and endostatin is particularly preferred for the detection of mildly elevated LV weight or early HF stages of abnormal MFS.

  Further preferred marker combinations for slight myocardial changes, abnormal MFS or mildly elevated LV weight or borderline LVH in early HF stage patient samples are FGF- for sensitive cardiac troponin, endostatin, and vitamin D Includes 23 ratios.

Marker levels of endostatin, NT-proBNP and cTnT in women or men with borderline or mildly elevated LV weight are shown in FIGS.
As can be obtained from the figure, several samples of subjects with slight myocardial changes are associated with low marker levels (less than the median indicated by the line). The median value relates to the marker level in the control group (stage 0 or A without slight myocardial changes). As can be further obtained from the graph, different sample subsets are detected by different markers. In particular, in female subjects, endostatin and NT-proBNP or endostatin and cTnT marker combinations are preferred to improve sensitivity.

Case Study A 75 year old female patient with class A heart failure has a BMI of less than 30 kg / m ² and hypertension. Troponin T and FGF-23 are determined in serum samples obtained from patients (using the above kit). The troponin T value is 7.1 pg / ml and the FGF-23 value is 93 RU / ml (relative units / ml, Immunotopics), which indicates that a diagnostic evaluation based on imaging should be performed. To confirm biomarker-based assessment, as an indirect estimate of increased LV filling pressure, left ventricular inflow and pulmonary venous Doppler-derived indices, and tissue Doppler images of lateral mitral annulus (E / A diagnostic test based on diastolic dysfunction imaging is performed using a Doppler-derived multiparametric algorithm, including e ′). The patient is diagnosed with diastolic dysfunction. This confirms that the detection of troponin T and FGF-23 allows a reliable assessment of whether the subject should be subjected to imaging based diagnostic assessment.

A 71 year old male patient with class A heart failure has a BMI of less than 30 kg / m ² and hypertension. Troponin T and FGF-23 are determined in serum samples obtained from patients. Troponin T values are less than 3.0 pg / ml and FGF-23 values are 59 RU / ml, indicating that diagnostic assessment based on imaging is not necessary. To confirm biomarker-based assessment, as an indirect estimate of increased LV filling pressure, left ventricular inflow and pulmonary venous Doppler-derived indices, and tissue Doppler images of lateral mitral annulus (E / A diagnostic test based on diastolic dysfunction imaging is performed using a Doppler-derived multiparametric algorithm, including e ′). The patient is diagnosed as not having diastolic dysfunction, thus confirming that the detection of troponin T allows a reliable assessment of whether the subject should be subjected to imaging-based diagnostic evaluation .

A 69 year old female patient with class A heart failure has a BMI of less than 30 kg / m ² and hypertension. Troponin T and FGF-23 are determined in serum samples obtained from patients. The troponin T value is 6.5 pg / ml and the FGF-23 value is 99 RU / ml, which is an indicator that a diagnostic evaluation based on imaging is necessary. Therefore, in order to confirm the evaluation based on the biomarker, the evaluation based on the imaging method is performed with respect to the contractile function. The LV contraction function is calculated at the mid-wall level (mid-wall shortening rate, MFS) using a modified oval model. In addition, LV weights are calculated according to recommendations from the American Echocardiographic Society and the European Echocardiology Association (EAE). The patient is diagnosed as having subnormal MFS and normal LV weight, and thus detection of troponin T allows a reliable assessment of whether the subject should be subjected to imaging-based diagnostic evaluation Is confirmed.

A 76 year old male patient with class A heart failure has a BMI of less than 30 kg / m ² and hypertension. Troponin T is determined in serum samples obtained from patients. Troponin T values are less than 3 pg / ml and FGF-23 values are 64 RU / ml, indicating that diagnostic assessment based on imaging is not necessary. Diagnose contractile function to confirm biomarker-based assessment. The LV contraction function is calculated at the mid-wall level (mid-wall shortening rate, MFS) using a modified oval model. In addition, LV weights are calculated according to recommendations from the American Echocardiographic Society and the European Echocardiology Association (EAE). The patient is diagnosed as having normal MFS and normal LV weight, which will allow a reliable assessment of whether troponin T detection should subject the subject to an imaging-based diagnostic assessment Confirm.

A 66 year old female patient with class A heart failure has a BMI of 40 kg / m ² and hypertension and diabetes. Troponin T is determined in serum samples obtained from patients. Troponin T values are less than 3.0 pg / ml, indicating that no diagnostic assessment based on imaging is required. The patient is told to return to follow-up after 12 months or if symptoms worsen. Twelve months later, patient comes to follow-up visit. Troponin T is determined in serum samples obtained from patients. Troponin T value is 3.5 pg / ml, indicating that diagnostic assessment based on imaging is necessary. Therefore, in order to confirm the evaluation based on troponin, a diagnostic test based on the imaging method of contractile function is performed. The LV contraction function is calculated at the mid-wall level (mid-wall shortening rate, MFS) using a modified oval model. In addition, LV weights are calculated according to recommendations from the American Echocardiographic Society and the European Echocardiology Association (EAE). The patient is diagnosed as having a subnormal MFS and normal LV weight, confirming that detection of troponin T allows a reliable assessment of whether the subject should be subjected to imaging-based diagnostic evaluation Is done.

Conclusions Conclusion: hs-cTnT and FGF-23 levels are elevated in elderly subjects with slight alterations in LV shape and function, and these levels identify an early intermediate phenotype in the progression of heart failure Can assist. In particular, hs-cTnT levels and FGF-23 level / vitamin D level ratios are elevated in elderly subjects with normal LV weight index and abnormal MFS.

  The widespread application of echocardiographic screening for systolic / diastolic dysfunction has been limited by considering cost / benefit. However, this disadvantage could be overcome by applying the method of the present invention. By determining the amount of cardiac troponin and / or FGF-23, it is possible to assess whether an imaging-based assessment of cardiac function is required. Therefore, unnecessary medical costs can be avoided by executing the method of the present invention.

  The detection of slight initial myocardial changes and / or early LVH can be improved by a combination of biomarkers selected from NT-proBNP, troponin T and endostatin. In addition, markers, mimecan, FGF23 / vitamin and GDF15 can be determined to detect slight initial myocardial changes.

  Imaging-based diagnostic evaluation for diagnosing diastolic or systolic dysfunction in hypertensive patients with normal left ventricular weight and not suffering from left ventricular hypertrophy by detection of the markers referred to herein Should be subjected to (preferably echocardiography) or can be stratified, where the increased level of the marker (s) relative to the respective reference level makes the patient useful for imaging-based diagnostic evaluation Indicates that it should be provided, and any other level of marker (s) indicates that the patient should not be subjected to imaging based diagnostic evaluation.

  In the current population of low-risk elderly, elevated levels of FGF-23 and low levels of vitamin D identify individuals with slight structural changes and predict all causes of death.

Claims (15)

A method for providing an analysis result for predicting the risk of death in a subject not suffering from heart failure and / or showing no obvious signs of heart failure comprising:
in a sample from a) the subject, determining the amount of IGFBP7, and b) the amount determined in step a), compared to a reference amount, whereby, for predicting the risk of mortality in the subject Provide analysis results ,
Said method comprising the steps.   The method of claim 1, wherein the subject has retained LVEF (left ventricular ejection fraction).   The method includes determining the amount of FGF-23 and further includes determining the amount of vitamin D, calculating a ratio between the amounts of FGF-23 and vitamin D, and comparing the ratio to a reference ratio. The method of claim 1 or 2.   4. The method of any one of claims 1-3, wherein step a) further comprises determining the amount of brain natriuretic peptide and / or cardiac troponin in a sample from the subject.   5. The method of any one of claims 1-4, wherein the subject does not suffer from heart failure.   6. The method of any one of claims 1-5, wherein the subject does not show obvious signs of heart failure. A subject who does not show obvious signs of heart failure suffers from heart failure classified as stage A or stage B according to the ACC / AHA classification, or the subject is healthy with respect to heart disease and disorder The method according to any one of 1 to 6 . Said sample, blood, serum or plasma sample, the method of any one of claims 1-7. Wherein the subject is a human, the method of any one of claims 1-8. Wherein the subject is female, the method of any one of claims 1-8. Use of a detection agent that specifically binds i ) IGFBP7 or ii ) IGFBP7 in a sample of a subject to predict the risk of death. Use of a detection agent that specifically binds i ) IGFBP7 and / or ii ) IGFBP7 in the manufacture of a composition for predicting the risk of death. Use according to claim 12 , wherein the composition is a pharmaceutical composition or a diagnostic composition. A device suitable for performing the method of any one of claims 1-10 , comprising:
a) an analyzer unit comprising a detection agent that specifically binds to IGFBP7, said unit being adapted to determine the amount of marker in the subject sample; and b) comparing the determined amount with a reference amount An analyzer unit for predicting the risk of death thereby comprising a database containing reference quantities and a computer-implemented algorithm for performing the comparison,
Including the device. 15. The device of claim 14 , wherein the analyzer unit in a) further comprises a detection agent that specifically binds to cardiac troponin and / or a detection agent that specifically binds to brain natriuretic peptide.

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